Therapeutic compounds and methods

ABSTRACT

This disclosure describes engineered compounds that engage NK cells and methods of using the compounds. Generally, the compound includes an NK engaging domain, a targeting domain that selectively binds to a target cell, and an NK activating domain operably linking the NK engaging domain and the targeting domain.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/237,835, filed Oct. 6, 2015, which is incorporated herein byreference.

GOVERNMENT FUNDING

This invention was made with government support under CA111412 andCA65493, awarded by the National Institutes of Health, and underCA36725, CA72669, and CA197292, awarded by the National CancerInstitute. The government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext file entitled “2016-10-06-SequenceListing_ST25.txt” having a sizeof 85 kilobytes and created on Oct. 6, 2016. The information containedin the Sequence Listing is incorporated by reference herein.

SUMMARY

This disclosure relates to the design, construction, and use oftrispecific killer engager (TriKE) molecules.

This disclosure describes, in one aspect, a molecule engineered topossess an NK engaging domain, an NK activating domain operably linkedto the NK engaging domain, and a targeting domain that selectively bindsto a target cell and is operably linked to the NK activating domain andthe NK engaging domain.

In some embodiments, the NK activating domain can include at least aportion of a cytokine.

In some embodiments, the NK engaging domain can include a moiety thatselectively binds to an NK cell. The moiety that selectively binds tothe NK cell can activate NK cells and/or block inhibition of NK cells.In some embodiments, the NK engaging domain can include an antibody or afragment thereof.

In some embodiments, the target cell can be a tumor cell or a cellinfected by a virus. In some embodiments, the targeting domain caninclude an antibody or a fragment thereof. In other embodiments, thetargeting domain can include a ligand or small molecule that selectivelybinds to the target cell.

In some embodiments, the molecule may be designed to include a secondtargeting domain, a second NK activating domain, or a second NK engagingdomain.

In another aspect, this disclosure describes a molecule engineered toinclude a T cell engaging domain, a T cell activating domain operablylinked to the T cell engaging domain, and a targeting domain thatselectively binds to a target cell and is operably linked to the T cellactivating domain and the T cell engaging domain.

In some embodiments, the T cell activating domain can include at least aportion of a cytokine.

In some embodiments, the T cell engaging domain can include a moietythat selectively binds to a T cell. The moiety that selectively binds tothe T cell can activate T cells and/or block inhibition of T cells. Insome embodiments, the T engaging domain can include an antibody or afragment thereof.

In some embodiments, the target cell can be a tumor cell or a cellinfected by a virus. In some embodiments, the targeting domain caninclude an antibody or a fragment thereof. In other embodiments, thetargeting domain can include a ligand or small molecule that selectivelybinds to the target cell.

In some embodiments, the molecule may be designed to include a secondtargeting domain, a second T cell activating domain, or a second T cellengaging domain.

In some embodiments of either aspect, the molecule can include aflanking sequence between any two of the domains summarized immediatelyabove. In some cases, the molecule can have more than one flankingsequence.

In another aspect, this disclosure describes a method that involvesadministering any embodiment of the engineered molecule summarized aboveto a subject in an amount effective to induce NK-mediated killing of thetarget cell or T-cell-mediated killing of the target cell, as may beappropriate for the particular molecule that is administered.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. 161533 trispecific killer engager (TriKE) (SEQ ID NO:1) elicitssuperior purification properties over 1633 bispecific killer engager(BiKE) (SEQ ID NO:2). (A) Schematic of coding region placement of theBiKE (left) and TriKE (right) domains in the pET expression vector. (B)Absorbance tracing for 1633 BiKE (left) eluted from the ion exchangecolumn as the first phase in drug purification using a three-stepelution protocol. The first peak eluted from the column represents theproduct. In the case of 161533 TriKE (right), the absorbance of peak 1nearly doubles indicating superior yield. Similar quantity of inclusionbodies were refolded and purified. All protein was removed from thecolumn. (C) SDS-PAGE gel and Coomasie Blue staining after a second steppurification over a size exclusion column. Densitometry analysisindicates that the product is over 95% pure.

FIG. 2. 161533 TriKE elicits superior NK cell function against targets.The release of the isotope chromium-51 (⁵¹Cr) is often used to measureNK cell function (killing). (A) Freshly isolated PBMCs were culturedwith chromium loaded HL-60 cells for four hours at E:T ratios of 20:1,6.6:1, and 2:1. Noted reagents were added at the beginning of co-cultureat a 20 nM concentration. Data is displayed as % NK cell cytolyticactivity. Given the number of conditions, significance only notedbetween 1633 and 161533 molecules. n=3. (B) To evaluate specificity of161533 TriKE, ⁵¹Cr release assay was performed against CD33⁻EpCAM⁺ HT29targets. EpCAM1533 TriKE was used as a positive control. n=2. (C) NKcells were enriched from normal donor PBMCs utilizing magnetic beads andplaced in culture with HL-60 targets (10:1) alone or in the presence of1633 BiKE or 161533 TriKE for 24 hours. At the end of the incubationsupernatants were taken from each of the cultures and frozen down forlater assessment of secreted IFNγ, TNFα, GM-CSF, and MIP1a throughLuminex multiplex assay (n=5). Points and Bars represent mean±SEM.

FIG. 3. The 161533 TriKE mediates NK cell proliferation and expansion.Post-transplant patient PBMCs were loaded with CELLTRACE proliferationdye (Thermo Fisher Scientific, Waltham, Mass.) and co-cultured withHL-60 Targets at a 5:1 (E:T) ratio for seven days in the presence of 50nM 1633 BiKE or 161533 TriKE. At the end of the incubation CD56⁺CD3⁻ NKcells were assessed for viability through Live/Dead Near IR staining.(A) Individual histogram and (B) pooled analysis of viability in the NKcell population treated with the 1633 BiKE (gray) or 161533 TriKE(black). (C) Proliferation was then assessed by CELLTRACE dilution inthe live CD56⁻CD3⁺ T cells (gray) and CD56⁺CD3⁻ NK cells (black) withinthe TriKE group. (D) Pooled analysis demonstrating % cells divided (top)and expansion index (bottom), which is a calculation of the foldexpansion within the population given the amount of CELLTRACE dilution.Individual dots represent separate post-transplant samples (n=8).

FIG. 4. 161533 TriKE potently rescues NK cell function inpost-transplant samples. Post-transplant patient PBMCs were thawed andrested overnight. The next night they were incubated with no drug, 1633BiKE (50 nM), or 161533 TriKE (50 nM). The next morning they were washedand given same treatment as the night before (to ensure that there areno issues with molecule internalization). (A) PBMCs with noted treatmentgroups were incubated with chromium loaded HL-60s for four hours and %cytolytic activity was calculated. Dot and bars denote mean±SEM (n=9).(B) Representative histograms and (C) pooled data of CD107a (leftpanels), IFNγ (center panels), and TNFα (right panels) expression onCD56⁺CD3⁻ NK cells after four-hour incubation with HL-60 targets. Dotsdenote individual patient samples (n=10).

FIG. 5. 161533 TriKE enhances NK cell function against primary AMLblasts compared to BiKE. Post-transplant patient PBMCs were thawed andrested overnight. The next night they were incubated with 1633 BiKE (50nM), or 161533 TriKE (50 nM). The next morning they were washed andgiven same treatment as the night before (to ensure that there are noissues with molecule internalization). Primary AML blasts from apheresisproducts of two separate patients were thawed and rested overnight.Treated post-transplant patient PBMCs (n=6) were incubated with the twodifferent primary AML blasts (n=12 total) for four hours and NK cellfunction was assessed by flow cytometry. NK function can be assessed bymeasuring lytic degranulation in the form of CD107a. (A) Representativehistograms denoting CD107a (left), IFNγ (center), and TNFα (right)expression on post-transplant patient NK cells treated with 1633 BiKE(gray) or 161533 TriKE (black) after four-hour incubation with primaryAML blasts. (B) Pooled data for CD107a (left), IFNγ (center), and TNFα(right) expression on post-transplant patient NK cells treated with 1633BiKE and 161533 TriKE and incubated with primary AML blasts. Each boxrepresents a separate post-transplant patient sample incubated againsttwo separate patient AML blast targets, denoted by filled and open boxes(n=12 total).

FIG. 6. 161533 TriKE limits HL-60 tumor growth in vivo better than 1633BiKE. HL-60-luc cells were injected iv (7.5×10⁵ cells/mouse) into NSGmice and three days later one million human NK cells activated overnightwith IL-15 were infused. The 1633 BiKE and 161533 TriKE groups receivedHL-60-luc and NK cells, while the control group only received HL-60-luccells. Drug (50 μg/kg) was administered MWF throughout the study. (A)Individual mouse photoluminscence at day 14 (top) and day 21 (bottom) ofstudy in a two-minute exposure (n=5 per treatment (unless mice died),representative of two separate experiments). (B) Quantification ofluminescence in mice from the three treatment groups at day 14 (left)and day 21 (right). Each dot represents a different mouse and barsdenote mean±SEM (n=5, representative of two separate experiments). (C)Blood was collected on day 20 from the mice in each of the experientialtreatment groups. Circulating CD56+CD3− human NK cells were quantifiedby flow cytometry. Events were collected over 60 seconds and the numberof human NK cell events was calculated. Representative dot plots areshown denoting the number of NK (CD56+.CD3⁻) cell events within theCD45+ gate. (D) Aggregate data demonstrating the number of human NK cellevents in each treatment group at day 20. Individual dots representdifferent mice and bars denote mean SEM (n=3 for HL-60-luc group [twomice died], n=5 for the 1633 BiKE and 161533 TriKE groups).

FIG. 7. The flanking sequences and the orientation of the TriKE moleculeboth influence its function. In order test the influence of the flankingsequences, a variant of 161533 (161533NL, SEQ ID NO:5) was constructedthat lacked the flanking sequences (PSGQAGAAASESLFVSNHAY, SEQ ID NO:3;and EASGGPE, SEQ ID NO:4) on either side of the IL-15 domain of the161533 construct. Freshly isolated PBMCs (containing 3.5% NK cells forthis example) from two independent donors (PB1 and PB2) were culturedwith chromium loaded HL-60 cells for four hours at an E:T ratio of 20:1.Noted reagents were added at the beginning of co-culture at a 20 nMconcentration. Data is displayed as % NK cell cytolytic activity. 161533reflects the construct that includes the modified IL-15 NK activatingdomain with the flanking sequences intact. The data indicate that theIL-15 NK activating domain with flanking sequences enhances TriKEfunction. To examine the effects of orientation, the construct wassynthesized with IL-15 on either the N terminus (151633; SEQ ID NO:6) orthe C terminus (163315; SEQ ID NO:7) and compared to wildtype 161533with IL-15 as a cross-linker. The data generated IL-15 in the center ofthe molecule optimizes NK cytolytic activity.

FIG. 8. 1615EpCAM TriKE (SEQ ID NO:8) elicits superior purificationproperties over EpCAM16 BiKE. (A) Schematic of placement of the codingregions for the TriKE (1615EpCAM) domains in the pET expression vector.(B) Schematic of placement of coding regions for the BiKE (16EpCAM)domains in the pET expression vector. (C) Absorbance tracing of theTriKE (1615EpCAM) eluted from the ion exchange column as the first phasein drug purification using a three-step elution protocol. The first peakeluted from the column represents the product. (D) Absorbance tracing ofthe BiKE (16EpCAM) eluted from the ion exchange column as the firstphase in drug purification using a three-step elution protocol. Thefirst peak eluted from the column represents the product, recovered at alower yield than the TriKE. (F) SDS-PAGE gel and Coomasie Blue stainingafter a second step purification (E) over a size exclusion column.Densitometry analysis indicates that the product is over 95% pure.

FIG. 9. Evaluation of the activity of the 1615EpCAM TriKE (SEQ ID NO:8)in chromium release assays. Freshly isolated Natural killer (NK)-cellswere added to HT-29 cells (human colorectal carcinoma cell line) withthe respective effector:target ratios as indicated. Donors were chosenwith naturally different levels of circulating NK cells (A) peripheralblood mononuclear cells (PBMC) with 3.8% NK cells, (B) PBMC with 6.4% NKcells, (C) 15% and (D)>80% NK cells enriched from PBMC. (E) Higherlevels of kill with 1615EpCAM correlated with donors with naturallyhigher levels of NK killing. In (E), only the curves for 1615EpCAM werecompared for the four donors, emphasizing the direct correlation betweenNK presence and cytolytic activity induced by the drug. (F) shows thatin the case of 16EpCAM, no such correlation exists.

FIG. 10. Lytic degranulation in different EpCAM expressing target cancercell lines. As mentioned, NK function can be measured by quantitatingCD107a expression as a measure of lytic degranulation. CD107a expressingcells were evaluated within the gated CD56⁺CD3⁻ NK cell population.Effector PBMCs were incubated with different EpCAM bearing target celllines including (A) BT-474, (B) SK-BR-3, (C) PC-3, (D) DU145, (E)UMSCC-11B, (F) NA, and (G) SKOV-1. TriKE added to effector and targetcells induced a higher percentages of CD107a-expressing cells comparedto controls and also compared to bispecific 16EpCAM. P-values wereestimated with one-way-ANOVA and presented with SD. *evaluation againstcontrols; # evaluation 1615EpCAM against EpCAM16.

FIG. 11. Proliferation capabilities of 1615EpCAM TriKE. (A) In order toevaluate NK cell expansion, peripheral blood mononuclear cells weretreated with 1615EpCAM TriKE or EpCAM16 BiKE. The discrete peaks in thehistograms mark successive generations of NK cells after cell divisionleading in a repetitive slight reduction of florescence intensity.Whereas NK cells show a typical proliferation pattern, T-cells do not.Shown is a representative of five independent experiments. (B) PBMCscells were co-cultured with the TriKE and the BiKE and NK cellproliferation was evaluated. Shown is a representative of fiveindependent experiments. (C) PBMCs cells were co-cultured with theTriKE, the BiKE, anti-CD16scFv [CD16], Interleukin (IL)-15, anti-EpCAMscFv [EpCAM] and DT2219, a targeted toxin comprised of Diphtheriaenterotoxin linked to anti-CD22 and anti-CD19 scFv. Evaluation of the NKcell Expansion Index showed a significantly (p<0.001) enhanced index inthe 1615EpCAM construct and with IL-15 alone, marked with *, (n=5). (D)Purified NK cells were exposed to the TriKE and the BiKE. After sevendays a reactive dye was used to differentiate alive and dead cells. Thereactive dye permeates the impaired membranes of dead cells, resultingin more intense staining (right peak) whereas failure to penetrate themembrane of live cells results in a weaker staining (left peak).

FIG. 12. Lytic degranulation and IFN-γ expression in HT-29 cells. Tostudy NK cell activity, CD107a-expressing cells were evaluated withinthe gated CD56⁺/CD3⁻ NK cell population. (A) Cells treated with the1615EpCAM TriKE (SEQ ID NO:8) showed precipitously elevateddegranulation of EpCAM-expressing HT-29 target cells, while controls didnot. E:T alone, E:T plus anti-EpCAM scFv devoid of 1615, E:T plusanti-CD16 alone, and E:T plus a combination of IL-12 and IL-18 (whichdoes not augment lytic degranulation) did not have any effect. (B) IFN-γproduction from the same CD56⁺/CD3⁻ NK cell population was analyzed.Only 1615EpCAM showed an enhanced percentage of IFN-γ-expressing cells.Values did not approach values seen with the IL12+IL18 combination thatis known to stimulate cytokine production at supraphysiologic levels.(C) No CD107a expression cells were observed when NK cells incubatedwith EpCAM-HL-60 myeloid leukemia cell targets were studied. (D) Onlythe E:T controls treated with IL12+IL18 showed precipitous expression ofIFN-γ.

FIG. 13. Schematic of the placement of the polynucleotide encoding1615EpCAM133 (SEQ ID NO:9) domains in the pET expression vector.Synthesis and assembly of the hybrid polynucleotide encoding1615EpCAM133 was accomplished using DNA shuffling and DNA ligationtechniques. The fully assembled coding region has, from the 5′ end tothe 3′ end, an NcoI restriction site; an ATG initiation codon; codingregions encoding the VH and VL regions of human CD16 (NM3E2) derivedfrom a phage display library, a 20 amino acid segment(PSGQAGAAASESLFVSNHAY; SEQ ID NO:3), modified IL-15, a seven amino acidsegment (EASGGPE; SEQ ID NO:4), the humanized anti-EPCAM scFv from theantibody MOC-31, a 15 amino acid mutated human IgG1 hinge region, andthe anti-CD133 scFv from clone 7; and finally a NotI restriction site.The resultant 2715 bp NcoI/NotI fragment polynucleotide was spliced intothe pET28c expression vector under control of anisopropyl-3-D-thiogalactopyranoside (IPTG) (FischerBiotech, Fair Lawn,N.J.) inducible T7promoter. DNA sequencing analysis (Biomedical GenomicsCenter, University of Minnesota, Minn., USA) was used to verify that thepolynucleotide was correct in sequence and had been cloned in frame.

FIG. 14. Activity of 1615EpCAM133. An extra scFv recognizing CD133expressed on cancer stem cells was added to 1615EpCAM to make1615EpCAM133 TetraKE. (A) and (B) show the activity of 1615EpCAM133evaluated with ⁵¹Cr release assays. Freshly isolated NK cells from twodonors (PT 1 and PT 2) were added to the human colorectal carcinoma cellline Caco-2 (CD133⁺, EpCAM⁺). Cells were co-cultured with targets atnoted effector to target (E:T) ratios for four hours and ⁵¹chromiumrelease was then evaluated. In (C) and (D), NK cells from two donorswere exposed to human colorectal carcinoma cell line HT-29 (EpCAM⁺,CD133⁻) and ⁵¹chromium release was measured in the same manner asdescribed above.

FIG. 15. Just like 1615EpCAM, 1615EpCAM133 shows enhanced expansion dueto the presence of IL-15. (A) Binding assays against HT-29 cells and (B)Caco-2 cells were performed using FITC-labeled 1615EpCAM133 TetraKE (200nM) competed with excess unlabeled noted scFvs (1000 nM). Experimentswere repeated with 200 nM of 1615EpCAM133 and a lower block with 500 nMof the scFv. Results were reproducible. (C) Purified NK cells werestained with CELLTRACE (Thermo Fisher Scientific, Waltham, Mass.) tomeasure proliferation and co-cultured with an anti-CD16 scFv [CD16],anti-CD133 scFv [CD133], 1615EpCAM133 TetraKE, DT2219 (mutateddiphtheria toxin linked to an anti-CD22 and an anti-CD19 scFv),anti-EpCAM scFv [EpCAM], EpCAM16 BiKE, or IL-15 [IL15] for seven days(n=5). Graph shows pooled data of the expansion index for each of thegroups. (D) Representative histogram of PBMCs stained with CELLTRACE dyeand co-cultured with 30 nM of 1615EpCAM133 TetraKE or EpCAM16 BiKE forseven days. (E) Representative histogram comparing proliferation onCD56⁺CD3⁻ NK cells with CD56⁻CD3⁺ T cells. (F) Representative histogramillustrating survival (by means of Live/Dead dye exclusion) of purifiedNK cells exposed to the 1615EpCAM133 TetraKE or EpCAM16 BiKE for sevendays. Dead cells display inclusion of the dye (high peak) while livecells exclude it (low peak). P-values were estimated with one-way-ANOVAand presented with standard deviation.

FIG. 16. Schematic of placement of the coding regions for 1615133 (SEQID NO:10) in the pET expression vector. The hybrid polynucleotideencoding 1615133 was synthesized using DNA shuffling and DNA ligationtechniques. The fully assembled polynucleotide has, from the 5′ end tothe 3′ end, a NcoI restriction site; an ATG start codon; coding regionsencoding anti-human CD16 scFv, a 20 amino acid segment(PSGQAGAAASESLFVSNHAY; SEQ ID NO:3), mutated human IL-15, a seven aminoacid linker (EASGGPE; SEQ ID NO:4), and anti-CD133 scFv; and a NotIrestriction site. The resultant 1884 base pair NcoI/NotI fragmentpolynucleotide was spliced into the pET28c expression vector undercontrol of an isopropyl-3-D-thiogalactopyranoside (IPTG) inducible T7promoter.

FIG. 17. ⁵¹Chromium release and binding of TriKE 1615133. (A, B) Forevaluation of activity, ⁵¹Chromium release assays using two donors wereperformed. 1615133 TriKE, 16133 BiKE, anti-CD16 scFv [CD16], oranti-CD133 scFv [CD133] was co-cultured with CD133⁺ Caco-2 cells andPBMCs at labeled E:T ratios. (C) PBMCs and Caco-2 cells were exposed todifferent TriKE concentrations (1 nM, 5 nM, 10 nM) and titered in theirE:T ratio (20:1, 6.6:1, 2.2:1, 0.75:1, 0.23:1, 0.08:1). (D) FITC-labeled1615133 TriKE was incubated at labeled concentrations with Caco-2 cells.In the same experiment, the same amount of FITC labeled 1615133 wasadded with 200 nM of a monomeric CD133 scFv for blocking.

FIG. 18. Expansion and survival. (A) Purified NK cells were exposed toanti-CD16 scFv (CD16), anti-CD133 scFv (CD133), 16133 BiKE, 1615133TriKE, DT2219 (a targeted toxin consisting of an anti-CD22 and anti-CD19scFv linked to a diphtheria toxin), or NCI-derived IL-15. Only the TriKEand IL-15 significantly increased proliferation (n=5). Graph showspooled data of the expansion index, calculated in Flowjo software, foreach of the groups. (B) Purified NK cells were exposed to 1615133 TriKEand 16133 BiKE and incubated for seven days. The representativehistogram illustrates a higher amount of live cells with the TriKEcompared to the BiKE construct without the IL-15 moiety. Significancewas estimated with one-way-ANOVA and presented with standard deviation.

FIG. 19. ⁵¹Chromium release assays were performed with several differentnew TriKEs to show that any scFv that targets cancer cells can be madeinto functional TriKEs. (A) EpCAM+CD133+NG2+ non-small cell lung cancerNCI-H460 cells plus NK cells were incubated with 1615EPCAM133 TriKE or1615NG2 TriKE (neuron glial antigen 2 or CSPG4). Both 1615NG2 and1615EpCAM133 had activity at several different E:T ratios (20:1, 10:1,and 5:1). (B) Mesothelin+EpCAM-CD133-NG2 MDA-435A melanoma cells wereincubated with 1615EPCAM TriKE (SEQ ID NO:8) or the 1615Meso TriKE (SEQID NO: 11) TriKE. Only 1615Meso had activity. (C) Mesothelin+NG2+ovarian cancer cells (Ovcar3 cells) were incubated with 1615NG2 TriKE or1615SS1 TriKE. 1615Meso and 1615NG2 had activity. (D) Raji cells werecultured with NK cells and studied in ⁵¹Cr release assays. TriKE16152219 (SEQ ID NO:12) simultaneously targets the B cell markers CD19and CD22. Only 16152219, 162219, and Rituximab killed the CD22+CD19+targets. The controls did not.

FIG. 20. TriKEs have been synthesized that work with IL-2 and stimulatethe expansion of T cells rather than NK cells. In order to determine ifother cytokines work in place of IL-15, CD3-IL-2-EpCAM (SEQ ID NO: 13)was constructed using the same flanking sequences on either side of IL-2that were used on either side of the IL-15 domain in the NK-activatingTriKE constructs. PBMCs were CELLTRACE (Thermo Fisher Scientific,Waltham, Mass.) labeled and placed in culture with no treatment (negcontrol), 1615EpCAM TriKE (pos control), CD3EpCAM BiTE, CD3-IL-2-EpCAMTriKE, 10 ng/ml IL-2, or 100 ng/ml IL-2. The CD3-IL2-EpCAM TriKEstimulated CD3⁺ T cells much greater than 1615EpCAM TriKE, CD3EpCAMBiTE, or IL-2 at either 10 ng/ml or 100 ng/ml.

FIG. 21. The CD3 portion of CD3-IL2-EpCAM was tested and intact. (A) and(B): the same anti-CD3 scFv was spliced to diphtheria toxin andincubated with CD3+ HPBMLT target cells. CD3-IL2-EpCAM was added to seeif it blocked the ability of the DT3 (CD3 targeted toxin) to killHPB-MLT cells. Blocking activity of CD3-IL-2-EpCAM was dose dependent inthe presence of 0.1 nM DT3 (A) and 1.0 nM DT3 (B), indicating that theCD3 moiety of CD3-IL2-EpCAM was intact. (C) and (D): the ability ofCD3-IL2-EpCAM to block the killing of negative control CD3− Raji cellsby DT2219 (anti-CD22 and CD19 targeted toxin).

FIG. 22. CD16 nanobody was derived from a published llama nanobody(GeneBank sequence EF561291). The CD16 nanobody was spliced to CD19 totest the ability of this CD16 engager to drive NK cell killing. (A) TheCD16 nanobody showed cytolytic NK activity similar to rituximab-mediatedkilling in a chromium release assay with CD19+Raji targets. (B) The CD16CDRs were cloned into a humanized camelid scaffold in order to generateHuEF91, a humanized CD16 engager. HuEF91 binding was equivalent toCD16scFv binding, indicating that the humanized HuEF91 did not hinderthe specificity of the molecule. (C) The llama161533 TriKE (SEQ IDNO:14) is capable of expanding NK cells.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immunesystem capable of immune surveillance. Like cytotoxic T cells, NK cellsdeliver a store of membrane penetrating and apoptosis-inducing granzymeand perforin granules. Unlike T cells, NK cells do not require antigenpriming and recognize targets by engaging activating receptors in theabsence of MHC recognition.

NK cells express CD16, an activation receptor that binds to the Fcportion of IgG antibodies and is involved in antibody-dependentcell-mediated cytotoxicity (ADCC). NK cells are regulated by IL-15,which can induce increased antigen-dependent cytotoxicity,lymphokine-activated killer activity, and/or mediate interferon (IFN),tumor-necrosis factor (TNF) and/or granulocyte-macrophagecolony-stimulating factor (GM-CSF) responses. All of theseIL-15-activated functions contribute to improved cancer defense.

Therapeutically, adoptive transfer of NK cells can, for example, induceremission in patients with refractory acute myeloid leukemia (AML) whencombined with lymphodepleting chemotherapy and IL-2 to stimulatesurvival and in vivo expansion of NK cells. This therapy can be limitedby lack of antigen specificity and IL-2-mediated induction of regulatoryT (Treg) cells that suppress NK cell proliferation and function.Generating a reagent that drives NK cell antigen specificity, expansion,and/or persistence, while bypassing the negative effects of Treginhibition, can enhance NK-cell-based immunotherapies.

This disclosure describes generating a tri-specific molecule thatincludes two domains capable of driving NK-cell-mediated killing oftumor cells (e.g., CD33⁺ tumor cells and/or EpCAM⁺ tumor cells) and anintramolecular NK activating domain capable of generating an NK cellself-sustaining signal. The tri-specific molecule can drive NK cellproliferation and/or enhance NK-cell-driven cytotoxicity against, forexample, HL-60 targets, cancer cells, or cancer cell-derived cell lines.

Bi-specific fusions have been made that incorporate an anti-humananti-CD16 scFv derived from a human phage display library technology(McCall et al., 1999. Mol Immunol. 36:433-445). NK cells mediateantibody-dependent cell-mediated cytotoxicity (ADCC) through the CD16(FcγRIII) receptor. Signaling through the CD16 receptor induces calciumfluxes and phosphorylation of ITAMs, triggering the release of lyticgranules and cytokines such as interferon (IFNγ) and tumor necrosisfactor (TNFα). A bi-specific molecule has been designed to trigger theCD16 receptor in conjunction with other targeting molecules (Gleason etal. Blood. 2014 (19):3016-26), a so-called bispecific killer engager(BiKE). With one scFv recognizing NK cells and a second scFv recognizinga tumor antigen, BiKEs can markedly enhance cytotoxic killing in varioushuman cancers. One exemplary BiKE targeted CD33 and enhanced NK cellresponses against acute myeloid leukemia (AML) and myelodyplasticsyndrome (MDS). MDS is a clonal heterogeneous stem cell disordercharacterized by normal or hypercellular bone marrow (BM) withperipheral blood (PB) cytopenias and an increased risk of progressing toAML.

NK cells are responsive to a variety of cytokines including, forexample, IL-15, which is involved in NK cell homeostasis, proliferation,survival, activation, and/or development. IL-15 and IL-2 share severalsignaling components, including the IL-2/IL-15Rβ (CD122) and the commongamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs,allowing for NK cell activation while bypassing Treg inhibition of theimmune response. Besides promoting NK cell homeostasis andproliferation, IL-15 can rescue NK cell functional defects that canoccur in the post-transplant setting. IL-15 also can stimulate CD8⁺ Tcell function, further enhancing its immunotherapeutic potential. Inaddition, based on pre-clinical studies, toxicity profiles of IL-15 maybe more favorable than IL-2 at low doses.

IL-15 plays a role in NK cell development homeostasis, proliferation,survival, and activation. IL-15 and 1-2 share several signalingcomponents including the IL-2/IL-15Rβ (CD122) and the common gamma chain(CD132). IL-15 also activates NK cells, and can restore functionaldefects in engrafting NK cells after hematopoietic stem celltransplantation (HSCT).

This disclosure describes, in one aspect, tri-specific killer engager(TriKE) molecules that generally include one or more NK cell engagerdomains (e.g., CD16, CD16+CD2, CD16+DNAM, CD16+NKp46), one or moretargeting domains (that target, e.g., a tumor cell or virally-infectedcell), and one or more cytokine NK activating domains (e.g., IL-15,IL-12, IL-18, 11-21, or other NK cell enhancing cytokine, chemokine,and/or activating molecule), with each domain operably linked to theother domains. As used herein, the term “operably linked” refers todirect or indirect covalent linking. Thus, two domains that are operablylinked may be directly covalently coupled to one another. Conversely,the two operably linked domains may be connected by mutual covalentlinking to an intervening moiety (e.g., and flanking sequence). Twodomains may be considered operably linked if, for example, they areseparated by the third domain, with or without one or more interveningflanking sequences.

The NK engaging domain can include any moiety that binds to and/oractivates an NK cell and/or any moiety that blocks inhibition of an NKcell. In some embodiments, the NK engaging domain can include anantibody that selectively binds to a component of the surface of an NKcell. In other embodiments, the NK engaging domain can include a ligandor small molecule that selectively binds to a component of the surfaceof an NK cell. As used herein, the term “selectively binds” refers tothe ability to differentiate between two or more alternatives such as,for example, having differential affinity, to any degree, for aparticular target. As used herein, “antibody” refers generally animmunoglobulin or a fragment thereof and thus encompasses a monoclonalantibody, a fragment thereof (e.g., scFv, Fab, F(ab′)₂, Fv or othermodified forms), a combination of monoclonal antibodies and/or fragmentsthereof, and/or a combination of polyclonal antibodies. Thus, forbrevity, reference to an antibody that selectively binds to a componentof the surface of an NK cell includes any antibody fragment thatexhibits the described binding character. Similarly, reference to aligand that selectively binds to a component of the surface of an NKcell includes any fragment of the ligand that exhibits the describedbinding character.

In some embodiments, the NK engaging domain can selectively bind to areceptor at least partially located at the surface of an NK cell. Incertain embodiments, the NK engaging domain can serve a function ofbinding an NK cell and thereby bring the NK into spatial proximity witha target to which the targeting domain-described in more detailbelow-selectively binds. In certain embodiments, however, the NKengaging domain can selectively bind to a receptor that activates the NKcell and, therefore, also possess an activating function. As describedabove, activation of the CD16 receptor can elicit antibody-dependentcell-mediated cytotoxicity. Thus, in certain embodiments, the NKengaging domain can include at least a portion of an anti-CD16 receptorantibody effective to selectively bind to the CD16 receptor. In otherembodiments, the NK engager cell domain may interrupt mechanisms thatinhibit NK cells. In such embodiments, the NK engager domain caninclude, for example, anti-PD1/PDL1, anti-NKG2A, anti-TIGIT,anti-killer-immunoglobulin receptor (KIR), and/or any other inhibitionblocking domain.

One can design the NK engaging domain to possess a desired degree of NKselectivity and, therefore, a desired immune engaging character. Forexample, CD16 has been identified as Fc receptors FcγRIIIa (CD16a) andFcγRIIIb (CD16b). These receptors bind to the Fc portion of IgGantibodies that then activates the NK cell for antibody-dependentcell-mediated cytotoxicity. Anti-CD16 antibodies selectively bind to NKcells, but also can bind to neutrophils. Anti-CD16a antibodiesselectively bind to NK cells, but do not bind to neutrophils. A TriKEembodiment that includes an NK engaging domain that includes ananti-CD16a antibody can bind to NK cells but not bind to neutrophils.Thus, in circumstances where one may want to engage NK cells but notengage neutrophils, one can design the NK engaging domain of the TriKEto include an anti-CD16a antibody.

While described herein in the context of various embodiments in whichthe NK engaging domain includes an anti-CD16 receptor scFv, the NKengaging domain can include any antibody or other ligand thatselectively binds to the CD16 receptor. Moreover, the NK engaging domaincan include an antibody or ligand that selectively binds to any NK cellreceptor such as, for example, the cell cytotoxicity receptor 2B4, lowaffinity Fc receptor CD16, killer immunoglobulin like receptors (KIR),CD2, NKG2A, TIGIT, NKG2C, LIR-1, and/or DNAM-1.

The targeting domain can include any moiety that selectively binds to anintended target such as, for example, a tumor cell, a target in thecancer stroma, a target on an inhibitory cell such as myeloid derivedsuppressor cells that are CD33+, or a target on a virally-infected cell.Thus, a targeting domain can include, for example, an anti-tumorantibody such as rituximab (anti-CD20), afutuzumab (anti-CD20),trastuzumab (anti-HER2/neu), pertuzumab (anti-HER2/neu), labetuzumab(anti-CEA), adecatumumab (anti-EpCAM), citatuzumab bogatox (anti-EpCAM),edrecolomab (anti-EpCAM), arcitumomab (anti-CEA), bevacizumab(anti-VEGF-A), cetuximab (anti-EGFR), nimotuzumab (anti-EGFR),panitumumab (anti-EGFR), zalutumumab (anti-EGFR), gemtuzumab ozogamicin(anti-CD33), lintuzumab (anti-CD33), etaracizumab (anti-integrinα_(v)β₃), intetumumab (anti-CD51), ipilimumab (anti-CD152), oregovomab(anti-CA-125), votumumab (anti-tumor antigen CTAA16.88), or pemtumumab(anti-MUC1), anti-CD19, anti-CD22, anti-CD133, anti-CD38anti-mesothelin, anti-ROR1, CSPG4, SS1, or IGFR1.

In other embodiments, the targeting domain can selectively bind to atarget on a cell infected by a virus such as, for example, anadenovirus, HIV, CMV, and/or HPV.

In certain particular embodiments, the targeting domain can include ananti-CD33 antibody. In other particular embodiments, the targetingdomain can include an anti-epithelial cell adhesion molecule (EpCAM)antibody.

The NK activating domain can include an amino acid sequence thatactivates NK cells, promotes sustaining NK cells, or otherwise promotesNK cell activity. The NK activating domain can be, or can be derivedfrom, one or more cytokines that can activate and/or sustain NK cells.As used herein, the term “derived from” refers to an amino acid fragmentof a cytokine (e.g., IL-15) that is sufficient to provide NK cellactivating and/or sustaining activity. In embodiments that include morethan one NK activating domain, the NK activating domains may be providedin series or in any other combination. Additionally, each cytokine-basedNK activating domain can include either the full amino acid sequence ofthe cytokine or may be an amino acid fragment, independent of the natureof other NK activating domains included in the TriKE molecule. Exemplarycytokines on which an NK activating domain may be based include, forexample, IL-15, IL-18, IL-12, and IL-21. Thus, while described in detailherein in the context of an exemplary model embodiment in which the NKactivating domain is derived from IL-15, a TriKE may be designed usingan NK activating domain that is, or is derived from, any suitablecytokine.

For brevity in this description, reference to an NK activating domain byidentifying the cytokine on which it is based includes both the fullamino acid sequence of the cytokine, any suitable amino acid fragment ofthe cytokine, and or a modified version of the cytokine that includesone or more amino acid substitutions. Thus, reference to an “IL-15” NKactivating domain includes an NK activating domain that includes thefull amino acid sequence of IL-15, an NK activating domain that includesa fragment of IL-15, or an NK activating domain such as, for example,IL-15N72D or IL-15N72A, that includes an amino acid substitutioncompared to the wild-type IL-15 amino acid sequence.

The use of an IL-15 NK activating domain in a TriKE can providesustained NK cell activity—as evidenced in a mouse model showing humanNK cells are dramatically elevated and cancer reduced-even after threeweeks. NK cells are activated in mice to produce an array of anti-cancerfactors and cytokines. Moreover, FIG. 1 shows that an IL-15 NKactivating domain somehow alters the chemistry of these molecules sothat they refold more easily and/or are recoverable in greater yield,thus rendering the TriKE molecules more suitable for clinical scale-up.

In some embodiments, the molecule can further include a flankingsequence that can link two of the above-described domains. In someembodiments, the presence of the flanking sequence can further increaseNK cell activation. One exemplary flanking sequence includes the 20amino acids of SEQ ID NO:3. Another exemplary flanking sequence includesthe seven amino acids of SEQ ID NO:4. Certain embodiments (e.g., the161533 TriKE, SEQ ID NO:1) can include more than one flanking sequence.As one example, SEQ ID NO: 1 includes the flanking sequence of SEQ IDNO:3 to link the NK engaging domain (e.g., anti-CD16 receptor scFv) withthe NK activating domain (e.g., IL-15). SEQ ID NO:1 also includes theflanking sequence of SEQ ID NO:4 to link the NK activating domain withthe targeting domain (e.g., anti-CD33 scFv). FIG. 7 shows datademonstrating that constructs that lack a flanking sequence exhibitreduced activity compared to constructs that possess the flankingsequence.

Synthesis and Purity of a 161533 TriKE

To create an exemplary model therapeutic TriKE that is antigen specificand self-sustains the NK cell response against leukemia, a humanmodified IL-15 cross-linker was introduced into the 1633 BiKE creating a161533 TriKE (FIG. 1A). The FPLC profile of the TriKE indicated a highyield product from a bacterial expression system that required refolding(FIG. 1B). The IL-15 NK activating domain reduced the isoelectric pointby two pH units, creating more favorable conditions for purification andenhancing yield. Despite purification beginning with identical amountsof inclusion bodies, the final yield of 161533 TriKE was twice the yieldof the comparable BiKE (1633, with no IL-15 NK activating domain),indicating more favorable purification dynamics. Products were >95% pureby SDS-PAGE gel analysis and Coomasie Blue Staining (FIG. 1C). To verifythat binding and specificity remained intact in the new TriKE molecule,selectivity was measured by direct binding and blocking flow cytometryassays against CD33⁺EpCAM⁻ HL-60 cells and CD33⁻ EpCAM⁺ HT-29 cells(Table 1 and Table 2).

TABLE 1 Binding of BiKE and TriKE Measured by Flow Cytometry ReagentCell Line Drug Amount (μg) % Positive Cells Unstained HL-60 — 0.1161533-FITC HL-60 10 63 161533-FITC HL-60 20 75 161533-FITC HL-60 40 78161533-FITC HT-29 10 4 EpCAM-FITC HL-60 20 1.4 EpCAM-FITC HT-29 2 100  1633-FITC HT-29 4 1   1633-FITC HL-60 4 62   1633-FITC HL-60 15 74    16-FITC HL-60 20 0.1     33-FITC HL-60 20 98

TABLE 2 Specificity Determined by Antigen Blockade Reagent Cell LineBlocking Agent % Positive Cells   1633-FITC HL-60 None 52   1633-FITCHL-60 anti-CD33 1 161533-FITC HL-60 None 85 161533-FITC HL-60 anti-CD334 161533-FITC HL-60 anti-CD45 73

161533 TriKE Increases NK Cell Function

To determine whether inclusion of IL-15 retained the ability ofbioengineered 1633 to mediate ADCC, 1633 and 161533 were compared in a4-hour chromium release assay where PBMCs from healthy donors weretested for their ability to kill CD33⁺ HL-60 targets (FIG. 2A). The161533 TriKE induced higher NK cell mediated killing than the BiKE,particularly at the 20:1 ratio (58.3±2.3% vs. 33±4%, P=0.0184). Controlsamples of anti-CD16 and anti-CD33 did not augment the response comparedto the untreated controls showing no activity of these components alone.To test specificity in a cytotoxic assay the 161533 TriKE was incubatedwith NK cells and CD33⁻ HT-29 target cells (FIG. 2B). The 161533 TriKEshowed no significant increases in killing of HT-29 cells when comparedto the no treatment control. To ensure that HT-29 target cells are notmerely more resistant as an explanation for specificity, the HT-29 cellswere incubated with a novel IL-15 TriKE containing an anti-EpCAM scFvinstead of anti-CD33. This TriKE robustly killed EpCAM⁺ HT-29 cells,highlighting the versatility of the IL-15 NK activating domain TriKEplatform against both hematologic malignancy and solid tumormalignancies.

Besides redirected cytotoxicity, another function of NK cells is toproduce cytokines and chemokines upon target cell recognition. To testif the TriKE enhances this process NK cells and HL-60 targets wereincubated without molecules, with 1633 BiKE, or with 161533 TriKE andsupernatants where collected after 24 hours and analyzed forinflammatory cytokines and chemokines (FIG. 2C). When compared to nodrug or the BiKE, the TriKE significantly induced IFNγ, TNFα, GM-CSF,and MIP-1α secretion. These data indicate that the IL-15 molecule in theTriKE can induce pro-inflammatory cytokine and chemokine secretion whichmay increase the anti-tumor activity of NK cells.

161533 TriKE Induces Survival and Expansion of Post-Transplant NK Cells

One therapeutic advantage of IL-15 is that it is involved in homeostasisand expansion of NK cells. Thus, the 161533 TriKE was tested to evaluateif these biological functions remain active within the TriKE molecule.To test this in a physiologically relevant context, earlypost-transplant patient samples were used. These samples provide asetting where NK cell reconstitution is needed to mediate anti-tumorgraft versus leukemia (GvL) responses. Evaluation of time points earlyafter transplant are of particular interest because defects in NK-cellsmediated target-cell-induced cytokine production at these same timepoints, which may account for early relapse (Foley et al., 2014. ImmunolRev 258(1):45-63). Post-Transplant Patient PBMCs (either day 100 [n=5]or earlier 20-44 [n=5] after transplant) were labeled with CELLTRACE dye(Thermo Fisher Scientific, Waltham, Mass.) to measure proliferation,incubated with HL-60 targets and either 1633 BiKE or 161533 TriKE forseven days, and then labeled with Live/Dead dye to measure NK cellsurvival. Within the PBMCs that were incubated with the 1633 BiKE, mostof the NK cells incorporated Live/Dead dye, indicating poor survival. Incontrast, patient PBMCs incubated with the 161533 TriKE supportedexcellent NK cell survival (FIGS. 3A and 3B; 96.9±0.5% vs. 21±5.4%;P<0.0001). To understand if the IL-15 moiety in the 161533 TriKE alsodrove proliferation, CELLTRACE dye dilution in the viable NK cellpopulation was evaluated. Unexpectedly, the 161533 TriKE induced robustand specific NK cell proliferation in the post-transplant patientsamples, with minimal proliferation of T cells (FIGS. 3C and 3D) in thesame sample (79.1±2.5% of the NK cells divided vs. 2.3±1.1% of the Tcells, P<0.0001). The NK cells also had a significantly higher expansionindex than total T cells (7.2±0.8% vs. 1.1±0.1%, P<0.0001), whichrepresents total fold expansion. This suggests that the activity ofIL-15 in the 161533 TriKE may be more NK cell specific as a result ofthe flanking scFv molecules in the construct. Moreover, incubating NKcells with the 161533 TriKE resulted in robust proliferation thatmirrored expansion mediated by a saturating concentration of IL-15.Thus, an IL-15 NK activating domain in the TriKE is functionally activeand capable of delivering a self-sustaining signal to healthy donor NKcells and/or can drive survival and proliferation of post-transplantpatient NK cells, a setting where NK cell reconstitution is defective.

161533 TriKE Rescues Defective NK Cell Function Early afterTransplantation

After allogeneic hematopoietic stem cell transplant, NK cells areincreased in number and respond to IL-12 and IL-18 stimulation butexhibit hyporesponsiveness for more than six months when exposed tocancer cell line targets. In this setting, short-term exposure toovernight incubation with IL-15 can rescue NK cell function against K562targets (Foley et al., 2011, Blood 118(10):2784-2792). Given thepotential clinical development of the exemplary TriKE 161533 molecule aspost-transplant immunotherapy, the TriKE and the comparable BiKE (1633)molecules were tested on post-transplant PBMCs from allogeneic siblinghematopoietic stem cell transplant recipients (FIG. 4). 1633 BiKE or161533 TriKE were incubated with NK cells overnight, to allow forfunctional recovery, and cells were incubated with HL-60 targets thenext morning and analyzed for function. Although incubation with the1633 BiKE resulted in a doubling of killing of HL-60 targets whencompared to just NK cells against HL-60 targets (18.9±2.1% versus9±1.5%, P<0.0001), incubation with the 161533 TriKE potently rescued NKcell-mediated cytotoxic function to a much great degree (52.1±3.5%) thanby the BiKE (FIG. 4A). The increase in cytotoxicity correlated well withincreased degranulation measured by CD107a expression (FIGS. 4B and 4Cleft panel). Compared to the BiKE, the TriKE potently rescued IFNγ(BiKE=2.6±0.5% vs. TriKE=21.7±4.4%, P=0.0012) and TNFα (BiKE=6.1±1% vs.TriKE=29.9±3.8%, P<0.0001) production (FIGS. 4B and 4C center and rightpanels). In all assays, the TriKE induced increased functionality whencompared to the BiKE. The magnitude of these changes clearly illustratethe immunotherapeutic potential of the 161533 TriKE in the earlypost-transplant setting.

161533 TriKE Increases NK Cell Function Against Primary AML Blasts

To compare the activity of 1633 BiKE and 161533 TriKE against primaryAML blasts, PBMCs from post-transplant patients were incubated withprimary AML blasts from two different patients (AML1 and AML2). CD107a,IFNγ and TNFα induction was reduced against the primary blasts comparedto HL-60 targets (FIG. 5 vs. FIG. 4). The decrease in function could beattributed in part to decreased expression of CD33 on primary blasts,but expression of inhibitory ligands or absence of activating receptorligands may also contribute to the decreased function. While nosignificant differences were seen in NK cell activation between AML1 andAML2 under the same conditions, PBMCs from post-transplant patientsamples incubated with the 161533 TriKE significantly (p<0.05) inducedgreater degranulation (CD107a) and cytokine production (IFNγ and TNFα)over the PBMCs incubated with the 1633 BiKE (FIG. 5A and FIG. 5B),suggesting that the combination of activation combined with IL-15 ispotent against primary AML targets. Taken together, these in vitro dataindicate that the 161533 TriKE can make NK cells antigen specificagainst primary tumor cells.

161533 TriKE Induces Enhanced In Vivo NK Cell Survival and Function

Comparing the in vivo activity of BiKE and TriKE required thedevelopment of a murine xenograft model that simultaneously accommodatedthe progression of CD33⁺ leukemia and human NK cells. HL-60 cellscontaining a luciferase reporter were injected intravenously (7.5×10⁵cells/mouse) and then three days later, 1 million human NK cellsactivated overnight with IL-15 were infused. FIG. 6 shows imaging datadepicting HL-60-luc tumor load in each of the treatment groups. Whilethe control group received only HL-60-luc cells, but no drug or NKcells, the 1633 BiKE and 161533 TriKE groups received HL-60-luc cellsand NK cells. Drug (50 μg/kg) was administered MTWThF throughout thestudy. On day 14 (FIG. 6B), BiKE and TriKE groups significantly (p<0.05)differed from the control group but not each other indicating that atearly time points both BiKE and TriKE impact tumor progressionsimilarly. On day 21, however, no significant differences could be foundbetween the no drug group and the BiKE group in surviving, although twomice from the no drug group died by this point. The TriKE group on theother hand significantly (p<0.05) differed from the control group, andat this time point also differed from the BiKE group, indicatingsuperior control of the HL-60 tumor burden at later stages with 161533TriKE therapy.

Given the NK cell survival and proliferation results noted in the invitro experiments from FIG. 3, the increased control of HL-60-luc tumorgenerated by the TriKE in vivo might be mediated, at least in part, byincreased maintenance and expansion of the transferred NK cellpopulation through IL-15 moiety in the 161533 TriKE molecule. Thus, micewere bled (20 μL) at day 20 and the number of NK cell (CD56⁺CD3⁻) eventsacquired during a fixed acquisition time (60 sec) was evaluated. Neitherthe control nor the 1633 BiKE treated animals showed significantevidence of circulating CD56⁺CD3⁻ human NK cells showing poor survivaland expansion under these conditions (FIGS. 6C and 6D). In markedcontrast, all of the 161533-TriKE-treated animals showed high levels ofhuman NK cells (4261±410.6 events). Mice treated with 161533 TriKE hadNK cell levels that were nearly 200-fold greater than BiKE NK levelsindicating a robust biological contribution of the IL-15 molecule as anNK activating domain within the TriKE molecule. Thus, use of an IL-15 NKactivating domain in the TriKE can reduce the need for therapy toinclude providing exogenous IL-15 to sustain NK cell numbers.

Flanking Sequences and Orientation Influence TriKE Activity

A variant of the 161533 construct was designed without the flankingsequences on either side of IL-15 in order to test the influence of theflanking sequences on the functionality of the molecule. The newvariant, identified herein as 161533NL (SEQ ID NO:5), was compared tothe 161533 construct (SEQ ID NO:1) in chromium release killing assays.FIG. 7 shows in two independent donors (PB-1 and PB2) that flankingsequences influence the activity of the TriKE. Orientation variants alsowere constructed with IL-15 in the N-terminus (151633; SEQ ID NO:6) andC-terminus (163315; SEQ ID NO:7) positions. FIG. 7 also shows that the161533 construct, which includes IL-15 in the center position as across-linker and possesses the flanking sequences, results in greater NKcell cytolytic activity. Without the flanking sequences, 161533NLexhibited cytotoxicity at an E:T ratio of 20:1 of 25% compared to theparental wildtype 161533 (57%), confirming that the flanking sequencesgenerally increase the NK cell cytolytic activity induced of TriKEconstructs.

1615EpCAM TriKE

To construct a self-sustaining hybrid immune engager, a 1615EpCAM TriKE(FIG. 8A, SEQ ID NO:8) was assembled by incorporating human IL-15 intothe EpCAM16 BiKE (FIG. 8B). The TriKE construct contains DNA fragmentsencoding the VH and VL regions of an anti-CD16 scFv, spliced to IL-15and then to the VH and VL regions of an anti-EpCAM scFv. The IL-15 DNAfragment is flanked on either side by a 20 amino acid (aa) segment (SEQID NO:3) and EASGGPE (SEQ ID NO:4). Absorbance tracing for 1615EpCAMTriKE and EpCAM16 BiKE eluted from the FFQ ion exchange column as thefirst phase in drug purification using a three-step elution protocol aredisplayed in FIGS. 8C and 8D, respectively. The first peak eluted fromthe column represents the product of interest. When a similar quantityof inclusion bodies were refolded and purified, yield was unexpectedlyimproved with the addition of the IL-15 cross linker. When compared tothe EpCAM16 BiKE, absorbance nearly tripled in the 1615EpCAM TriKEindicating superior yield. SDS-PAGE gel and Coomasie Blue staining showpurity after both ion exchange and size exclusion column purifications(FIGS. 8E and 8F) resulting in a product that is over 90% pure with asize of about 68860 kDa. Thus, just as observed with the 161533 TriKE(SEQ ID NO:1), incorporating IL-15 directly into a hybrid TriKE conferssuperior purification properties in comparison to the correspondingBiKE—in this case, EpCAM16—lacking IL-15.

1615EpCAM TriKE Induces Chromium-51 Release

To determine the functional activity of 1615EpCAM, its killing abilitywas measured in standard ⁵¹chromium release assays (FIG. 9). Todetermine the effect of incorporating IL-15 into the EpCAM16 scaffold,NK-cell-mediated cytotoxicity was evaluated in a wide range of donorshaving different NK cell contents. Freshly isolated PBMCs were added toHT-29 cells at Effector (E):Target (T) ratios of 20:1, 6.6:1, and 2.2:1,generating cytolytic curves. The engineered reagents were added at theconcentration of 30 nM (maximum effective dose after titrationexperiments). Donors with 3.8%, 6.4%, 15%, and enriched NK cells >80%(as determined by flow cytometry) showed that the IL-15 componentgenerally improves the killing capabilities of NK cells (FIGS. 9A, 9B,9C, and 9D, respectively). In FIG. 9E, only the donor curves for1615EpCAM were graphed, emphasizing a direct correlation betweenincreasing NK presence and cytolytic activity. FIG. 9F shows that forEpCAM16, no such correlation exists. Due to baseline variation,reproducibility was ensured by repetition with different donors.Together, the data indicate that greater the number of NK cells in theassay, the greater the observed NK cytolytic activity.

1615EpCAM TriKE Induces Lytic Degranulation and IFN-γ Expression inVarious Cell Lines

To determine whether other EpCAM-expressing target cell lines inducedsimilar 1615EpCAM TriKE-mediated NK cell activation as the HT-29 targetline, NK cell function was tested on a variety of targets in conjunctionwith different drug treatments. Breast cancer (FIGS. 10A and 10B),prostate cancer (FIGS. 10C and 10D), head and neck cancer (FIGS. 10E and10F), and ovarian cancer cell lines (FIG. 10G) were studied. All EpCAM⁺carcinoma lines treated with 1615EpCAM (SEQ ID NO:8) inducedsignificantly elevated NK cell degranulation (p<0.001) when compared tovarious controls including E:T alone, E:T plus IL-15, E:T plus CD16CD133(an irrelevant BiKE), and E plus IL-12/IL-18. The E:T plus EpCAM16 BiKEalso demonstrated marked percentages of cells expressing CD107a andcytotoxic activity since the BiKE possesses cytotoxic activity but lacksthe ability to expand. Thus, in all cases, the values observed using theEpCAM16 BiKE were significantly less than values observed for 1615EpCAM(p<0.001).

1615EpCAM TriKE Induces NK Cell Proliferation

The ability of the 1615EpCAM TriKE (SEQ ID NO:8) to induce proliferationin NK cells is shown in FIG. 11. When donor PBMCs were exposed to theTriKE, NK cells but not T cells showed a proliferation-specific patternas measured by flow cytometry (FIG. 11A). The results were identical inthree of the four donors. When exposed to the TriKE, NK cells undergo amore robust proliferation than T cells. FIG. 11B shows a directcomparison of NK-proliferation induced by the EpCAM16 BiKE and the1615EpCAM TriKE. The TriKE induces proliferation and expansion, but theBiKE does not. To exclude the possibility of other factors that couldinduce NK cell proliferation, PBMCs were exposed to TriKE, BiKE,anti-CD16 scFv alone, IL-15 alone, anti-EpCAM scFv alone, or DT2219 (atargeted toxin comprising diphtheria toxin, linked to an anti-CD22 scFvand anti-CD19 scFv). Only TriKE-treated groups and IL-15-treated groupsdisplayed significant NK cell proliferation, as indicted by the changesin expansion index (FIG. 11C), which reflects the fold expansion of thecells. The group stimulated with the TriKE showed higher NK cellsurvival while the group exposed to the BiKE contained predominantlydead cells, as confirmed with forward/side scatter flow cytometry (FIG.11D) and trypan blue staining. These data indicate that besidesincreasing priming of the cells, the IL-15 moiety in the 1615EpCAM TriKEalso induces expansion and maintenance of the NK cells.

1615EpCAM TriKE Induces Lytic Degranulation and IFN-γ Expression AgainstHT-29 Target Cells

To study lytic degranulation as a parameter of NK cell activity, CD107aexpression was measured within a CD56⁺/CD3⁻ NK cell population incubatedwith EpCAM-expressing HT-29 targets. Cells incubated with EpCAM16 BiKEshowed elevated CD107a expression when compared with effectors alone,effectors plus targets without drug, or effectors plus targets withanti-EpCAM scFv. The 1615EpCAM TriKE induced significantly more CD107aexpression than the BiKE (FIG. 12A). The 1615EpCAM also inducedsignificantly elevated degranulation when compared to an extensive panelof controls (including E:T alone, E:T plus anti-EpCAM scFv devoid of1615, E:T plus anti-CD16 ScFv alone, CD2219, and E:T plus a combinationof IL-12 and IL-18) that did not have any effect. Two different sourcesof stand-alone IL-15, when combined with E:T, also failed to enhancelytic degranulation (IL-15 self, linker protein; IL-15 NCI, NCIderived). IFN-γ production also was enhanced in the 1615EpCAMTriKE-treated NK cells when compared to NK cells treated with the BiKEalone or the BiKE plus IL-15, indicating the biological ability of theIL-15 moiety within the TriKE to induce priming for cytokine secretion(FIG. 12B). As before, an extensive panel of controls was tested againstthe TriKE, in which only IL-12/IL-18 supraphysiologic stimulationoutperformed the TriKE.

In FIG. 12C, no CD107a expression was observed when NK cells wereincubated with control EpCAM⁻ HL-60 myeloid leukemia cells. No elevationof IFN-γ expression was observed as expected with negative control HL-60targets, except for control cells treated with IL-12/IL-18, showing theIFN-γ assay was working (FIG. 1D).

1615EpCAM133 Induces Chromium-51 Release

The design of the engineered tetraspecific 1615EpCAM133 (SEQ ID NO:9) isshown in FIG. 13. 1615EpCAM133 activity was evaluated with chromiumrelease assays in order to measure NK cell killing. The assay wasperformed using Caco-2 (CD133⁺, EpCAM) and HT-29 (CD133⁻, EpCAM⁺)targets and freshly isolated NK cells of two donors (PT1 and PT2) foreach cancer cell line, with no antibody (No Ab), anti-CD16 scFv,anti-CD133 scFv, anti-EpCAM scFv, IL-15 alone, and the EpCAM16 BiKE runas controls. In all donors and in both cancer cell types, 1615EpCAM133showed superior killing at increasing E:T ratios (FIG. 14A-D). Controlsshowed minimal activity. These data show that targeting two differentmoieties on the tumor cell is possible. In this case, one is a broaderepithelial marker target (EpCAM) and the other is the more specificanti-cancer stem cell target CD133.

1615EpCAM133 Induces NK Cell Proliferation

The ability of 1615EpCAM133 to selectively bind is shown in FIGS. 15Aand 15B. The ability of the IL-15 moiety within the molecule to induceproliferation and survival is shown in FIG. 15C-F. Purified NK cellswere exposed to an anti-CD16 scFv, anti-CD133 scFv, 1615EpCAM133, DT2219(mutated diphtheria toxin linked to an anti-CD22 and an anti-CD19 scFv),anti-EpCAM scFv, EpCAM16 BiKE, or IL-15 alone (NCI). Expansion index,which determines overall expansion of the culture, showed significantlyenhanced expansion in the 1615EpCAM133 and in the IL-15 groups(p<0.001), (FIG. 15C). To compare the ability of the 11-15 linker toinduce proliferation, PBMCs or purified NK cells were cultured afterstaining with a reactive dye and exposed to the EpCAM16 BiKE or the1615EpCAM133 tetraspecific molecule. After incubation, flow cytometrywas performed on gated CD56⁺CD3⁻ cells to evaluate NK cells and onCD56⁻CD3⁺ cells to evaluate T-cells. In FIG. 15D, only NK cells treatedwith 1615EpCAM133 showed substantial proliferation. Treatment with theEpCAM16 BiKE did not. The ability to induce specific proliferation to NKcell is shown in FIG. 15E; T cells did not proliferate after exposure to1615EpCAM133.

To study the ability of 1615EpCAM133 to enhance survival of NK cells,purified NK cells were co-cultured for seven days and treated with1615EpCAM133 or EpCAM16 BiKE. After live-dead staining via flowcytometry, a much higher percentages of live NK cells were seen in the1615EpCAM133 group (FIG. 15F).

1615133 Induces Chromium-51 Release

The design of the engineered 1615133 is shown in FIG. 16. In order toevaluate functional activity of the 1615133 TriKE, standard ⁵¹Chromiumrelease assays were performed. To determine the effect of incorporatingIL-15 into the 16133 scaffold, the cytotoxicity was evaluated using NKcells of two separate donors and Caco-2 tumor targets at different E:Tratios (20:1, 10:1, and 5:1) and compared activity between 1615133TriKE, 16133 BiKE, anti-CD16 scFv, anti-CD133 scFv, and no drugtreatment (FIGS. 17A and 17B). Killing of Caco-2 targets was elevated inthe TriKE compared to controls. Dose dependent titration of the 1615133TriKE (1 nM, 5 nM, and 10 nM) with a broader spectrum of E:T ratios(20:1, 6.6:1, 2.2:1, 0.7:1, 0.23:1, and 0.08:1) showed highest impact ofthe drug activity at higher doses (FIG. 17C). To evaluate specificity ofbinding, flow-cytometry-based fluorescence intensity was measured afterincubating Caco-2 cells with FITC-labeled 1615133 TriKE in differentconcentrations (1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 200 nM, or 500 nM).When an unlabeled anti-CD133 scFv (200 nM) was added along with the1615133 TriKE, binding was potently reduced (FIG. 17D), indicating thatthe 1615133 TriKE binds to target cells specifically through interactionwith CD133. Together, these data indicated that the ADCC mediated by theTriKE is antigen directed.

1615133 Induces NK Cell Proliferation

The proliferation induced by 1615133 (SEQ ID NO:10) was measured byCELLTRACE dye dilution in the viable NK and T cell populations. Whendonor PBMCs were exposed to 1615133 TriKE or 16133 BiKE, only the TriKEgroup induced proliferation (FIG. 18A). Importantly, comparison to othercontrol agents including anti-CD16 scFv, anti-CD133 scFv, DT2219 (atargeted toxin consisting of an anti-CD22 and anti-CD19 scFv linked to adiphtheria toxin), and NCI derived IL-15 showed that only the 1615133TriKE and NCI IL-15 induced proliferation. To show the potential of1615133 TriKE to induce prolonged survival, purified NK cells wereincubated for seven days with 1615133 or 16133 BiKE. A reactive dye wasused to quantify cell death in the different treatment groups. The TriKEgroup showed a greater amount of live cells, which do not incorporatethe reactive dye, compared to the BiKE (FIG. 18B). Together, the resultsindicate that the IL-15 present in the 1615133 TriKE induces NK cellproliferation and prolonged their survival.

TriKEs Generally Induce Chromium-51 Release

51 Chromium release assays were performed with several different TriKEsto show that any scFv that targets cancer cells can be incorporated intoa functional TriKE. Non-small cell lung cancer cells (NCI-H460) cellswere incubated with the 1615EPCAM133 TriKE (SEQ ID NO:9) or the 1615NG2TriKE. Both 1615NG2 and 1615EpCAM133 had activity at several differentE:T ratios (20:1, 10:1, and 5:1). FIG. 19B shows melanoma cells wereincubated with the 1615EPCAM133 TriKE. Mesothelin+EpCAM-NG2 MDA-435Amelanoma cells were incubated with 1615EPCAM TriKE, or the 1615MesoTriKE (SEQ ID NO: 11). Only 1615Meso had activity. Ovarian cancer cells(Ovcar3 cells) were incubated with the 1615NG2 TriKE or the 1615MesoTriKE. Both TriKEs induced NK cytolytic activity. Also, an anti-leukemicTriKE was made recognizing the leukemia markers CD19 and CD22. 16152219TriKE was tested on CD22+CD19+ Raji cells and killed them very well (aswell as rituximab). Together, these data show that any scFv can beinserted into the generalized TriKE structural platform of 1615X and theresulting TriKE can direct NK cells to respond to the scFv target andexpand. Additional exemplary TriKE molecules are listed in Table 3.

TABLE 3 Exemplary TriKE molecules TriKE molecule Target(s) ADCC*Expansion** Activation*** 161533 CD33 + + + 1615EpCAM EpCAM + + +1615EpCAM133 EpCAM/ +/+ +/+ +/+ CD133 1615133 CD133 + + + 1615NG2NG2 + + + 1615Meso mesothelin + + + 1615ROR-1 ROR-1 + + + 16a1538CD38 + + + 1615IGF-1 IGF1 + + + 1615Her2 Her2/neu + + + 16152219CD22/CD19 +/+ +/+ +/+ Llama161533 CD33 + + + 1615HIV HIV + + + *ADCC orcytotoxic activity enhanced over 30% by TriKE platform **Expansion:TriKE enhances expansion of NK cells, BiKE does not. ***Activation:TriKE enhances the production of various anti-cancer cytokines includingINFγ and TNFα.

Several TriKEs have been produced and tested in an identical manner, buttarget different cancer markers. CD33 or Siglec-3 (sialic acid bindingIg-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67) is a transmembranereceptor expressed on cells of myeloid lineage. It is usually consideredmyeloid-specific. EpCAM, epithelial cell adhesion molecule, is atransmembrane glycoprotein mediating Ca²⁺-independent homotypiccell-cell adhesion in epithelia. CD133, also known as prominin-1, is aglycoprotein that in humans is encoded by the PROM1 gene and a member ofpentaspan transmembrane glycoproteins (5-transmembrane, 5-TM), whichspecifically localize to cellular protrusions. NG2 is chondroitinsulfate proteoglycan 4, also known as melanoma-associated chondroitinsulfate proteoglycan (MCSP) or neuron-glial antigen 2 (NG2). Itrepresents an integral membrane chondroitin sulfate proteoglycanexpressed by human malignant melanoma cells. Mesothelin is a 40 kDaprotein present on normal mesothelial cells and overexpressed in severalhuman tumors, including mesothelioma and ovarian and pancreaticadenocarcinoma. ROR-1 is a receptor tyrosine kinase that modulatesneurite growth. It is a type I membrane protein belonging to the RORsubfamily of cell surface receptors and is currently under investigationfor its role in the metastasis of cancer cells. HER2 is a member of thehuman epidermal growth factor receptor (HER/EGFR/ERBB) family. CD38(cluster of differentiation 38), also known as cyclic ADP ribosehydrolase, is a glycoprotein found on the surface of many immune cells.IGF-1 is insulin-like growth factor 1 (IGF-1), also called somatomedinC. IGF-1 is a protein that in humans is encoded by the IGF1 gene andassociated with breast cancer. The human immunodeficiency virus (HIV) isa lentivirus (a subgroup of retrovirus) that causes HIV infection andover time acquired immunodeficiency syndrome (AIDS) and Kaposi Sarcoma.

CD3-IL2-EpCAM TriKE Selectively Boosts Proliferation of T Cells

TriKEs have been synthesized that work with IL-2 in place of IL-15 withthe same flanking sequences. These stimulate the expansion of T cellsrather than NK cells. The T-cell-directed TriKE CD3-IL-2-EpCAM (SEQ IDNO: 13) was synthesized and tested for its ability to stimulate T cellsrather than NK cells (FIG. 20). IL-2 is known to be a better stimulantof T cell proliferation than IL-15. PBMCs were CELLTRACE (Thermo FisherScientific, Waltham, Mass.) labeled and placed in culture. The histogramshows that CD3-IL-2-EpCAM drove robust proliferation of T cells, but notNK cells. CD3-IL-2-EpCAM showed better stimulation than any other agenttested including 1615EpCAM TriKE (pos control), CD3EpCAM BiTE,CD3-IL-2-EpCAM, IL-2, or no treatment. These data indicate that IL-2works in the same manner as IL-15 when used as a cross-linker on thisplatform.

Thus, this disclosure describes the design and use of a tri-specifickiller engager (TriKE) capable of creating immunologic synapses betweenNK cells and a target. A CD33⁺ myeloid target was used as a model targetfor a model TriKE that included an anti-CD16 antibody as a model NKengaging domain, an anti-CD33 antibody as a model targeting domain thattargeted the CD33⁺ myeloid target, a model IL-15-based NK activatingdomain, and flanking sequences on either side of the NK activatingdomain linking the NK activating domain to the remaining domains. Theflanking sequences are PSGQAGAAASESLFVSNHAY (SEQ ID NO:3) upstream ofthe NK activating domain and EASGGPE (SEQ ID NO:4) downstream of the NKactivating domain. The flanking sequences influence the functionalactivity of the TriKE molecules and represents an entirely unexpectedfinding.

One exemplary model TriKE (161533, SEQ ID NO:1) exhibited increasedfunction to a comparable bi-specific killer engager (1633 BiKE, SEQ IDNO:2) in cytotoxicity, CD107a degranulation, and cytokine productionassays of NK-cell-mediated responses against HL-60 targets. The activityof the exemplary model TriKE in a physiologic context was evaluatedusing patient NK cells collected early after allogeneic stem celltransplantation, a context where NK cell function is defective. Comparedto the 1633 BiKE, the TriKE containing an IL-15 NK activating domaininduced NK cell but not T cell survival and proliferation. The exemplarymodel TriKE molecule also induced the hyporesponsive patient NK cells tomediate potent responses against primary acute myeloid leukemia targets.Lastly, the exemplary model TriKE molecule exhibited superior anti-tumoractivity compared to the comparable BiKE, and induced in vivopersistence and survival of human NK cells for at least three weeks in axenogeneic model using HL-60-Luc and human NK cells.

The data presented herein establish the utility of the exemplary TriKEmolecule and provides the foundation for the design and construction ofalternative TriKE molecules. As described in detail above, a TriKEmolecule may be designed using any suitable NK engaging domain and/orany suitable targeting domain.

The exemplary 161533 TriKE (SEQ ID NO:1) was used as a model becauseCD16 is expressed on the surface of NK cells. Thus, an scFv thatselectively binds to NK cells can be used as an NK cell engaging domain.CD33 is expressed on AML acute myeloid leukemia cells (a common form ofadult leukemia), but is also found on myelodysplastic cells that maysignal a predisposition to AML. Thus, and anti-CD33 scFv can be used asa targeting domain.

In an alternative embodiment, however, an anti-EpCAM antibody can beused in the targeting domain of a TriKE. EpCAM is an epithelial cancermarker expressed on most types of carcinoma including, for example,lung, breast, colorectal, prostate, pancreatic, GI, renal, and ovariancancer. Data showing that a TriKE (1615EpCAM, SEQ ID NO:8) that includesan anti-EpCAM scFv enhances killing of colorectal cancer cells indicatesthat any suitable targeting domain (e.g., any suitable scFv) can beincluded in a TriKE based on the anti-CD16/IL15 platform with similarsuccess. The 1615EpCAM TriKE includes an anti-CD16 scFv as the NKengaging domain, and IL-15 NK activating domain, and the anti-EpCAM scFvas the targeting domain.

In yet another alternative embodiment, CD38 is known to be expressed onmultiple myeloma cells. Data showing that a TriKE (16a1538, SEQ IDNO:16) that includes an anti-CD38 scFv enhances killing of multiplemyeloma cells in vitro further indicates the general modularity of theTriKE platform. The 16a1538 TriKE includes an anti-CD16a scFv as the NKengaging domain, an IL-15 NK activating domain, and an anti-CD38 scFv asthe targeting domain. Finally, one can create a TetraKE (tetramer) bydesigning the molecule to include a second targeting domain. Forexample, one can design a TetraKE to include an anti-CD133 scFv (SEQ IDNO:17) to, for example, the 1615EpCAM TriKE (SEQ ID NO:8) to form anexemplary TetraKE (1615EpCAM133, SEQ ID NO:9). CD133 is an establishedmarker on cancer stem cells. Cancer stem cells represent the smallpopulation of stem cells in a tumor that are responsible for tumorinitiation, renewal, and chemotherapy resistance.

In some embodiments this disclosure describes an immune engager thatsimultaneously mediates ADCC and provides a self-sustaining signalinducing NK effector cell expansion and maintenance. Although a BiKEthat includes anti-CD16 scFv spliced to anti-EpCAM scFv promotedformation of an immune synapse between NK effector cells andEpCAM-expressing carcinoma cells that resulted in cytotoxicdegranulation culminating in ADCC of the target cells, both cytotoxicactivity and NK longevity can benefit by the addition of a costimulatorysignal that enhances effector cell expansion directly at the site ofimmune engagement. In some embodiments, this costimulatory signal isprovided by adding an agent well-suited for expanding NK cells. Forexample, to facilitate selective NK expansion, IL-15 was cross-linkedinto EpCAM16 BiKE. As shown in Example 2, the molecular addition ofIL-15 to an immune engager can mediated NK proliferation, can producesustained ADCC activity, and can improve lytic degranulation andcytokine secretion of the immune engager.

In some embodiments, the NK cell engager can involve the use of ahumanized CD16 engager derived from an animal nanobody. While an scFvhas a heavy variable chain component and a light variable chaincomponent joined by a linker, a nanobody consists of a single monomericvariable chain—i.e., a variable heavy chin or a variable lightchain—that is capable of specifically engaging a target. A nanobody maybe derived from an antibody of any suitable animal such as, for example,a camelid (e.g., a llama or camel) or a cartilaginous fish. A nanobodycan provide superior physical stability, an ability to bind deepgrooves, and increased production yields compared to larger antibodyfragments.

In one exemplary embodiment, a nanobody-based NK engager molecule caninvolve a humanized CD16 nanobody derived from a published llamananobody (GeneBank sequence EF561291; Behar et al., 2008. Protein EngDes Sel. 21(1):1-10), termed EF91. Llama EF91 was initially constructedinto a BiKE containing CD19 to test the ability of this CD16 engager todrive NK cell activation. It showed functionality similar torituximab-mediated killing in a chromium release assay with Raji targets(FIG. 22A). Upon confirming functionality of the molecule, the CDRs werecloned into a humanized camelid scaffold (Vincke et al., 2009. J BiolChem. 284(5):3273-3284) to humanize the CD16 engager, now termed HuEF91.The binding of HuEF91 is shown in FIG. 22B and is equivalent to bindingobserved using a standard CD16 scFv, indicating that incorporating thellama nanobody variable heavy chain into the humanized backbone has nothindered the specificity of the molecule. The use HuEF91 as an NKengager in the TriKE molecules described herein can increase drug yield,increase stability, and/or increase NK-cell-mediated ADCC efficacy.

In some embodiments, an immune engager as described herein can be usedto stimulate a patient's own immune system to eliminate tumor cells.Although studies show that T cells, genetically modified to expresschimeric antigen receptors (CARs), are powerful clinical mediators ofanti-tumor activity, production of T-CARs is costly and complex. Otherdisadvantages include the risk of cytokine toxicity and long-termpersistence of T-CARs resulting in interaction with healthy tissue orneoplastic transformation. As described herein, a tri-specific killerengager can serve as a mediator of ADCC and can expand NK cells withoutthe need of extracorporal genetic modification and gene therapy,providing a potential advantage over the T-CAR system. Because theimmune engager is rapidly cleared, the response cannot be indefinitelysustained, perhaps reducing the risk of cytokine toxicity of the immuneengagers compared to T-CARs.

In some embodiments, a tri-specific killer engager includes a cytokine.In some embodiments, a tri-specific killer engager preferably includesIL-15. IL-15 does not induce T_(regs) and IL-15 is a regulator of NKcells. In addition to improving activation and cytotoxicity, IL-15 canregulate and initiate anti-apoptotic and proliferative signals on NKcells, leading to enhanced NK cell expansion and survival. Thesecharacteristics can be beneficial during the use of the tri-specifickiller engager in the treatment against cancer. In some embodiments,including IL-15 in the tri-specific killer engager can mediate directeddelivery of the TriKE to the NK/Target cell synapse, potentially causingIL-15 to accumulate at a tumor site more effectively than systemicIL-15.

In some embodiments, a tri-specific killer engager preferably includesIL-15, anti-CD16 scFv, and anti-EpCAM scFv (1615EpCAM TriKE). In someembodiments, IL-15 acts as a crosslinker between the anti-CD16 scFv andthe anti-EpCAM scFv.

In some embodiments, the immune engager increases the secretion of animmune cell-mediated cytokine. In some embodiments, the cytokinesecretion is preferably antigen specific. In some embodiments, thiscytokine can include IFN-γ, GM-CSF, IL-6, IL-8, and/or TNF-α. In someembodiments, this cytokine production is preferably at physiologiclevels. In some embodiments, this cytokine production is at a levellower than the level observed in an IL-12/IL-18 stimulated NK cell(Papadakis et al., 2004. J Immunol. 172:7002-7007). As shown in Example2, measuring hallmark inflammatory cytokines including GM-CSF, IL-6,IL-8, TNF-α using a cytokine Luminex analysis demonstrates astatistically significant difference in GM-CSF secretion between BiKEand TriKE but no difference in the secretion of other cytokines.

In some embodiments, the immune engager increases proliferation of alymphocyte. The lymphocyte can include, for example, an NK cell, a γδ-Tcell, and/or, a CD8 T cell.

Just as the 1615EpCAM133 TetraKE molecule includes more than onetargeting domain, one can design a TetraKE, or larger molecule, thatincludes more than one NK cell engager domain and/or more than one NKactivating domain.

In another aspect, this disclosure describes methods of killing a targetcell in a subject. Generally, the method includes administering to thesubject a TriKE molecule in an amount effective to induce NK-mediatedkilling of the target cells. “Treat” or variations thereof refer toreducing, limiting progression, ameliorating, or resolving, to anyextent, the symptoms or signs related to a condition. As used herein,“ameliorate” refers to any reduction in the extent, severity, frequency,and/or likelihood of a symptom or clinical sign characteristic of aparticular condition; “symptom” refers to any subjective evidence ofdisease or of a patient's condition; and “sign” or “clinical sign”refers to an objective physical finding relating to a particularcondition capable of being found by one other than the patient.

A “treatment” may be therapeutic or prophylactic. “Therapeutic” andvariations thereof refer to a treatment that ameliorates one or moreexisting symptoms or clinical signs associated with a condition.“Prophylactic” and variations thereof refer to a treatment that limits,to any extent, the development and/or appearance of a symptom orclinical sign of a condition. Generally, a “therapeutic” treatment isinitiated after the condition manifests in a subject, while“prophylactic” treatment is initiated before a condition manifests in asubject. Thus, in certain embodiments, the method can involveprophylactic treatment of a subject at risk of developing a condition.“At risk” refers to a subject that may or may not actually possess thedescribed risk. Thus, for example, a subject “at risk” for developing aspecified condition is a subject that possesses one or more indicia ofincreased risk of having, or developing, the specified conditioncompared to individuals who lack the one or more indicia, regardless ofthe whether the subject manifests any symptom or clinical sign of havingor developing the condition. Exemplary indicia of a condition caninclude, for example, genetic predisposition, ancestry, age, sex,geographical location, lifestyle, or medical history. Treatment may alsobe continued after symptoms have resolved, for example to prevent ordelay their recurrence.

In some cases, the treatment can involve administering the TriKEmolecule to a subject so that the TriKE molecule can stimulateendogenous NK cells in vivo. Using a TriKE molecule as a part of an invivo can make NK cells antigen specific with simultaneousco-stimulation, enhancement of survival, and expansion, which may beantigen specific. In other cases, the TriKE can be used in vitro as anadjuvant to NK cell adoptive transfer therapy.

In another aspect, the TriKE can be designed to activate T cells ratherthan NK cells. In this aspect, the TriKE can generally include one ormore T cell engaging domains, one or more T cell activating domains, andone or more targeting domain (that target, e.g., a tumor cell orvirally-infected cell), and one or more T ell activating domains (e.g.,IL-2 or other T cell enhancing cytokine, chemokine, and/or activatingmolecule), with each domain operably linked to the other domains.

The T cell engaging domain can include any moiety that binds to and/oractivates a T cell and/or any moiety that blocks inhibition of a T cell.In some embodiments, the T cell engaging domain can include an antibodyor fragment thereof that selectively binds to a component of the surfaceof a T cell. In other embodiments, the T cell engaging domain caninclude a ligand or small molecule that selectively binds to a componentof the surface of a T cell.

In some embodiments, the T cell engaging domain can selectively bind toa receptor at least partially located at the surface of a T cell. Incertain embodiments, the T cell engaging domain can serve a function ofbinding a T cell and thereby bring the T cell into spatial proximitywith a target to which the targeting domain-described in more detailbelow-selectively binds. In certain embodiments, however, the T cellengaging domain can selectively bind to a receptor that activates the Tcell and, therefore, also possess an activating function.

While described herein in the context of various embodiments in whichthe T cell engaging domain includes an anti-CD3 receptor scFv, the Tcell engaging domain can include any antibody or other ligand thatselectively binds to the CD3 receptor. Moreover, the T cell engagingdomain can include an antibody or ligand that selectively binds to any Tcell receptor such as, for example, an anti-CD4 antibody, an anti-CD8antibody, an anti-LFA-1 antibody, an anti-LFA-2 antibody, an anti-CTLA4antibody, an anti-TCR antibody, an anti-CD28 antibody, an anti-CD25antibody, an anti-PD1 antibody, PD-IL, B7-1, B7-2, MHC molecules, CD80,CD86, B7H, an anti-SLAM antibody, or an anti-BTLA antibody.

The targeting domain can include any moiety that selectively binds to anintended target such as, for example, a tumor cell, a target in thecancer stroma, a target on an inhibitory cell such as myeloid derivedsuppressor cells that are CD33+, or a target on a virally-infected cell.Thus, a targeting domain can include, for example, any one of thetargeting domains described above in the context of NK-activating TriKEmolecules.

The T cell activating domain can include an amino acid sequence thatactivates T cells, promotes sustaining T cells, or otherwise promotes Tcell activity. The T cell activating domain can be, or can be derivedfrom, one or more cytokines that can activate and/or sustain T cells. Asused herein, the term “derived from” refers to an amino acid fragment ofa cytokine (e.g., IL-2) that is sufficient to provide T cell activatingand/or sustaining activity. In embodiments that include more than one Tactivating domain, the T activating domains may be provided in series orin any other combination. Additionally, each cytokine-based T activatingdomain can include either the full amino acid sequence of the cytokineor may be an amino acid fragment, independent of the nature of other Tcell activating domains included in the TriKE molecule.

Exemplary cytokines on which a T cell activating domain may be basedinclude, for example, IL-2 or any cytokine of the IL-2 family thatshares a chain with the IL-2 receptor such as, for example, IL-15, IL-4,IL-7, IL-9, IL-21, and IL-13. Thus, while described in detail herein inthe context of an exemplary model embodiment in which the T cellactivating domain is derived from IL-2, a TriKE may be designed using aT cell activating domain that is, or is derived from, any suitablecytokine.

For brevity in this description, reference to a T cell activating domainby identifying the cytokine on which it is based includes both the fullamino acid sequence of the cytokine and any suitable amino acid fragmentof the cytokine. Thus, reference to an “IL-2” T cell activating domainincludes a T cell activating domain that includes the full amino acidsequence of IL-2 or an T cell activating domain that includes a fragmentof IL-2. In some embodiments, therefore, the T cell activating domaincan include the amino acid sequence of SEQ ID NO: 18.

In another aspect, this disclosure describes methods of killing a targetcell in a subject. Generally, the method includes administering to thesubject a TriKE molecule in an amount effective to induceT-cell-mediated killing of the target cells. Here again, the treatmentmay be therapeutic or prophylactic as described above in the context ofmethods that involve the use of an NK-activating TriKE.

Accordingly, a TriKE molecule-whether an NK-activating TriKE or aT-cell-activating TriKE—may be administered before, during, or after thesubject first exhibits a symptom or clinical sign of the condition.Treatment initiated before the subject first exhibits a symptom orclinical sign associated with the condition may result in decreasing thelikelihood that the subject experiences clinical evidence of thecondition compared to a subject to which the TriKE molecule is notadministered, decreasing the severity of symptoms and/or clinical signsof the condition, and/or completely resolving the condition. Treatmentinitiated after the subject first exhibits a symptom or clinical signassociated with the condition may result in decreasing the severity ofsymptoms and/or clinical signs of the condition compared to a subject towhich the composition is not administered, and/or completely resolvingthe condition.

The TriKE molecule can be any embodiment of the TriKE molecule describedabove having a targeting domain that selectively binds to an appropriatetarget cell population. In some cases, the target cell can include atumor cell so that the method can involve treating cancer associatedwith the tumor cells. Thus, in some embodiments, the method can includeameliorating at least one symptom or clinical sign of the tumor.

In embodiments in which the target cell includes a tumor cell, themethod can further include surgically resecting the tumor and/orreducing the size of the tumor through chemical (e.g., chemotherapeutic)and/or radiation therapy. Exemplary tumors that may be treated includetumors associated with prostate cancer, lung cancer, colon cancer,rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renalcancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterinecancer, thyroid cancer, skin cancer, head and neck cancer, cervicalcancer, ovarian cancer and/or hematopoietic cancer.

In various embodiments, the TriKE targeting domain can include apolypeptide that selectively binds to, for example, EGFR, HER2/neuEpCAM, CSPG4, HSPG2, IGF-1, CD38, CD19, CD20, CD22, CD30, CD52, CD33,ROR-1, UPAR, VEGFR, CD33, LIV-1, SGN-CD70A, CD70, IL-3, IL-4R, CD133,mesothelin, the epithelial-mesenchymal transition (EMT), TRAIL, CD38,CD45, CD74, CD23, or cancer viral markers such as HIV.

As used herein, a “subject” can be any animal such as, for example, amammal (e.g., dog, cat, horse, cow, sheep, goat, monkey, etc.). Incertain embodiments, the subject can be a human.

A TriKE molecule described herein may be formulated with apharmaceutically acceptable carrier. As used herein, “carrier” includesany solvent, dispersion medium, vehicle, coating, diluent,antibacterial, and/or antifungal agent, isotonic agent, absorptiondelaying agent, buffer, carrier solution, suspension, colloid, and thelike. The use of such media and/or agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions. As usedherein, “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with a TriKE molecule withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

A TriKE molecule may therefore be formulated into a pharmaceuticalcomposition. The pharmaceutical composition may be formulated in avariety of forms adapted to a preferred route of administration. Thus, acomposition can be administered via known routes including, for example,oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous,intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g.,intranasal, intrapulmonary, intramammary, intravaginal, intrauterine,intradermal, transcutaneous, rectally, etc.). A pharmaceuticalcomposition can be administered to a mucosal surface, such as byadministration to, for example, the nasal or respiratory mucosa (e.g.,by spray or aerosol). A composition also can be administered via asustained or delayed release.

Thus, a TriKE molecule may be provided in any suitable form includingbut not limited to a solution, a suspension, an emulsion, a spray, anaerosol, or any form of mixture. The composition may be delivered informulation with any pharmaceutically acceptable excipient, carrier, orvehicle. For example, the formulation may be delivered in a conventionaltopical dosage form such as, for example, a cream, an ointment, anaerosol formulation, a non-aerosol spray, a gel, a lotion, and the like.The formulation may further include one or more additives including suchas, for example, an adjuvant, a skin penetration enhancer, a colorant, afragrance, a flavoring, a moisturizer, a thickener, and the like.

A formulation may be conveniently presented in unit dosage form and maybe prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing a TriKE molecule into association with acarrier that constitutes one or more accessory ingredients. In general,a formulation may be prepared by uniformly and/or intimately bringingthe active molecule into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into the desired formulations.

The amount of TriKE molecule administered can vary depending on variousfactors including, but not limited to, the specific TriKE molecule beingused, the weight, physical condition, and/or age of the subject, and/orthe route of administration. Thus, the absolute weight of TriKE moleculeincluded in a given unit dosage form can vary widely, and depends uponfactors such as the species, age, weight and physical condition of thesubject, and/or the method of administration. Accordingly, it is notpractical to set forth generally the amount that constitutes an amountof TriKE molecule effective for all possible applications. Those ofordinary skill in the art, however, can readily determine theappropriate amount with due consideration of such factors.

In some embodiments, the method can include administering sufficientTriKE molecule to provide a dose of, for example, from about 100 ng/kgto about 50 mg/kg to the subject, although in some embodiments themethods may be performed by administering TriKE molecule in a doseoutside this range. In some of these embodiments, the method includesadministering sufficient TriKE molecule to provide a dose of from about10 μg/kg to about 5 mg/kg to the subject, for example, a dose of fromabout 100 μg/kg to about 1 mg/kg.

Alternatively, the dose may be calculated using actual body weightobtained just prior to the beginning of a treatment course. For thedosages calculated in this way, body surface area (m²) is calculatedprior to the beginning of the treatment course using the Dubois method:m²=(wt kg^(0.425)×height cm^(0.725))×0.007184.

In some embodiments, the method can include administering sufficientTriKE molecule to provide a dose of, for example, from about 0.01 mg/m²to about 10 mg/m².

In some embodiments, a TriKE molecule may be administered, for example,from a single dose to multiple doses per week, although in someembodiments the method can be performed by administering a TriKEmolecule at a frequency outside this range. In certain embodiments, aTriKE molecule may be administered from about once per month to aboutfive times per week.

In some embodiments, the method further includes administering one ormore additional therapeutic agents. The one or more additionaltherapeutic agents may be administered before, after, and/or coincidentto the administration of a TriKE molecule. A TriKE molecule and theadditional therapeutic agents may be co-administered. As used herein,“co-administered” refers to two or more components of a combinationadministered so that the therapeutic or prophylactic effects of thecombination can be greater than the therapeutic or prophylactic effectsof either component administered alone. Two components may beco-administered simultaneously or sequentially. Simultaneouslyco-administered components may be provided in one or more pharmaceuticalcompositions. Sequential co-administration of two or more componentsincludes cases in which the components are administered so that eachcomponent can be present at the treatment site at the same time.Alternatively, sequential co-administration of two components caninclude cases in which at least one component has been cleared from atreatment site, but at least one cellular effect of administering thecomponent (e.g., cytokine production, activation of a certain cellpopulation, etc.) persists at the treatment site until one or moreadditional components are administered to the treatment site. Thus, aco-administered combination can, in certain circumstances, includecomponents that never exist in a chemical mixture with one another. Inother embodiments, the TriKE molecule and the additional therapeuticagent may be administered as part of a mixture or cocktail. In someaspects, the administration of TriKE molecule may allow for theeffectiveness of a lower dosage of other therapeutic modalities whencompared to the administration of the other therapeutic agent or agentsalone, thereby decreasing the likelihood, severity, and/or extent of thetoxicity observed when a higher dose of the other therapeutic agent oragents is administered.

Exemplary additional therapeutic agents include altretamine, amsacrine,L-asparaginase, colaspase, bleomycin, busulfan, capecitabine,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin,daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide,fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine,hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan,mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine,oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed,temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine,vincristine, vindesine, and vinorelbine.

In some embodiments, of the method can include administering sufficientTriKE molecule as described herein and administering the at least oneadditional therapeutic agent demonstrate therapeutic synergy. In someaspects of the methods of the present invention, a measurement ofresponse to treatment observed after administering both a TriKE moleculeas described herein and the additional therapeutic agent is improvedover the same measurement of response to treatment observed afteradministering either the TriKE molecule or the additional therapeuticagent alone. In some embodiments, an additional therapeutic agent caninclude an additional agent that targets EpCAM including, for example,an EpCAM specific monoclonal antibody, such as, for example,Catumaxomab, a monoclonal hybrid antibody targeting EpCAM and CD3.

In the preceding description and following claims, the term “and/or”means one or all of the listed elements or a combination of any two ormore of the listed elements; the terms “comprises,” “comprising,” andvariations thereof are to be construed as open ended—i.e., additionalelements or steps are optional and may or may not be present; unlessotherwise specified, “a,” “an,” “the,” and “at least one” are usedinterchangeably and mean one or more than one; and the recitations ofnumerical ranges by endpoints include all numbers subsumed within thatrange (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Cell Isolation, Patients and Samples

PBMCs from age-matched normal donors were isolated from adult bloodobtained from Memorial Blood Center (Minneapolis, Minn.) bycentrifugation using a Histopaque gradient (Sigma-Aldrich, St. Louis,Mo.) and cryopreserved. For the post-transplant patient sample study,matched sibling donor allogeneic hematopoietic cell transplant sampleswere used from an immune reconstitution tissue bank. Recipient PBMCswere collected at either day 100 [n=5] or earlier (day 20-44) [n=5]after transplant and cryopreserved for future use. All samples wereobtained after informed consent, using guidelines approved by theCommittee on the Use of Human Subjects in Research at the University ofMinnesota in accordance with the Declaration of Helsinki.

Cell Lines

HL-60, a CD33⁺ human acute promyelocytic leukemia cell line (ATCC,Manassas, Va.), was cultured in Iscove's medium (Invitrogen, Carlsbad,Calif.) supplemented with 20% FBS (Gibco-Invitrogen) and 100 U/mLpenicillin and 100 U/mL streptomycin (Invitrogen, Carlsbad, Calif.) at37° C. and 5% CO₂. The control human colorectal carcinoma cell lineHT-29 (ATCC) was cultured at 37° C. with 5% CO₂ in Dulbecco's ModifiedEagle's Medium (DMEM), high glucose (Invitrogen, Carlsbad, Calif.)supplemented with 10% FBS and 100 U/mL penicillin and 100 U/mLstreptomycin.

Construction, Expression and Purification of BiKEs and TriKEs

The hybrid polynucleotide encoding 161533 (SEQ ID NO: 1) was synthesizedusing DNA shuffling and DNA ligation techniques (Vallera et al., 2013Cancer Biother Radiopharm 4:274-482; Vallera et al., 2009. Leuk Res33(9):1233-1242). Coding regions for the VL and VH of each scFv werelinked by a fragment encoding G4S linker. In its final configuration,the 161533 NcoI/XhoI polynucleotide has a start codon followed first bycoding regions for anti-human CD16 scFv (McCall et al., 1999. MolImmunol. 7:433-445), a 20 amino acid flanking polypeptide(PSGQAGAAASESLFVSNHAY; SEQ ID NO:3), human IL-15N72D, a seven amino acidflanking polypeptide (EASGGPE; SEQ ID NO:4), and then the anti-CD33scFv. The polynucleotide was spliced into the pET28c expression vectorand inclusion bodies expressed. DNA-sequencing analysis (BiomedicalGenomics Center, University of Minnesota) was used to verify that theploynucleotide was correct in sequence and cloned in frame.

The same components were used to construct a hybrid polynucleotideencoding 163315 (SEQ ID NO:7), except that the order of the componentswas CD16scFv, the flanking polypeptide PSGQAGAAASESLFVSNHAY (SEQ IDNO:3), anti-CD33 scFv, the flanking polypeptide EASGGPE (SEQ ID NO:4),then human IL-15.

Plasmid was transformed into the Escherichia coli strain BL21(DE3)(EMD,Madison Wis.). Bacteria were grown in 600 ml Luria Broth supplementedwith 100 μg/ml kanamycin in a 2 L flask at 37° C. with shaking.Expression of the hybrid polynucleotide was induced by the addition ofisopropyl-b-D-thiogalactopyranoside (IPTG, FisherBiotech Fair Lawn,N.J.). Two hours after induction, the bacteria were harvested bycentrifugation. The cell pellets were suspended and homogenized using apolytron homogenizer. After sonication and centrifugation, the pelletswere extracted with 0.3% sodium Deoxycholate, 5% Triton X-100, 10%Glycerin, 50 mM Tris, 50 mM NaCl, 5 mM EDTA, pH 8.0 and inclusion bodiesextensively washed to remove endotoxin.

The proteins were refolded using a sodium N-lauroyl-sarcosine (SLS) airoxidation method modified from a previously reported procedure forisolating scFv (Vallera et al., 2005. Leuk Res 29(3):331-341). Refolded161533 was purified by FPLC ion exchange chromatography (Q SepharoseFast Flow, Sigma, St. Louis, Mo.) using a stepwise gradient from 0.2 Mto 0.5 M NaCl in 20 mM Tris-HCl, pH 9.0 over four column volumes.

Flow Cytometry

Cells were immunophenotyped with the following fluorescent-labeledmonoclonal antibodies (mAb) against: PE-Cy7-conjugated CD56 (HCD56;BioLegend, Inc., San Diego, Calif.), ECD/PE-CF594-conjugated CD3 (UCHT1;Beckman Coulter, Brea, Calif.), APC-Cy7-conjugated CD16 (3G8; BioLegend,Inc.), Pacific Blue-conjugated CD45 (HI30; BioLegend, Inc.),PerCP-Cy5.5/FITC-conjugated anti-human CD107a (LAMP-1) (H4A3; BioLegend,Inc.), Pacific Blue/BV421-conjugated anti-human IFN-γ (4S.B3; BioLegend,Inc.), FITC/Alexa Fluor 647-conjugated TNF-α (MAb11; BioLegend, Inc.),FITC/PE-conjugated CD33 (P67.6; BD Biosciences), and APC-conjugated CD45(HI30; BioLegend, Inc.), FITC-conjugated EpCAM; (BioLegend, Inc.).Phenotypic acquisition of cells was performed on the LSRII (BDBiosciences) and analyzed with FlowJo software (Tree Star Inc., Ashland,Oreg.).

CD107a and IFNγ/TNFα Functional Flow Assay

Post-transplant patient PBMCs or primary AML blasts were thawed andplaced in RPMI-10 overnight. The next night, the PBMCs were incubatedwith 50 nM 1633 BiKE or 161533 TriKE. The next morning cells were washedand another round of 50 nM 1633 BiKE or 161533 TriKE was added toaddress any possible issues with molecule internalization. HL-60 Targetsor primary AML blasts were added immediately after to generate a 5:1effector to target ratio. PBMCs, HL-60 targets or primary AML blasts,and BiKE or TriKE molecules were co-cultured for four hours and CD107aexpression and intracellular IFN-γ and TNF-α production were evaluatedas previously described (Vallera et al., 2013 Cancer Biother Radiopharm4:274-282).

Proliferation Assay

PBMCs from post-transplant patients (day 100 or earlier [day 20-44])were labeled with CELLTRACE Violet Cell Proliferation Dye (Thermo FisherScientific, Waltham, Mass.), per manufacturer's protocol, placed inculture medium with HL-60 target cells at 5:1 (E:T) ratio, and treatedwith 50 nM 1633 BiKE or 161533 TriKE. Cells were then harvested sevendays later and analyzed for viability, through Live/Dead staining, andproliferation, through dilution of CELLTRACE, in the NK cell (CD56⁺CD3⁻)population.

51-Chromium Release Cytotoxicity Assay

Cytotoxicity was evaluated by 4-hour ⁵¹Cr-release assays. Briefly,resting PBMC from normal donors treated with the 1633 BiKE (10 μg/mL),scFvCD16 control reagent (10 μg/mL) or no reagent were co-cultured forfour hours with ⁵¹Cr-labeled or HL-60 targets at varying E:T ratios. Forpost-transplant study PBMCs cells were with HL-60 targets at a 20:1(E:T) ratio in the presence of 50 nM 1633 BiKE or 50 nM 161533 TriKE.⁵¹Cr release was measured by a gamma scintillation counter (PerkinElmer, Walthman, Mass.) and specific target lysis was determined(Vallera et al., 2013. Cancer Biother Radiopharm 4:274-282).

In vivo Mouse Study and Imaging

NSG mice (n=5/group) were conditioned with 275 cGy and injected IV with0.75×10⁵ HL-60-luc S4 subcultured for tumor invasiveness. Drug treatmentwas begun on day 3. A single course of treatment consisted of anintraperitoneal (IP) injection of 20 μg of drug given every day for aweek (MTWThF) and mice were treated for three weeks. The control groupreceived no NK cells while the 1633 BiKE and the 161533 TriKE groupsreceived 1×10⁶ NK cells, calculated from a CD3/CD19 magneticallydepleted product, three days after injection of the HL-60-luc cells. TheHL-60-luc cells contain a luciferase reporter, allowing for imaging ofthe mice each week to determine their bioluminescent activity andmonitor cancer leukemia progression as described previously (Waldron etal., 2011. Mol Cancer Ther 10(10):1829-1838.). Briefly, mice wereinjected with 100 μl of 30 mg/ml luciferin substrate 10 minutes prior toimaging and then anesthetized via inhalation of isoflurane gas. The micewere then imaged using the Xenogen Ivis 100 imaging system and analyzedwith Living Image 2.5 software (Xenogen Corporation, Hopkington Mass.).On day 20, all the animals were bled and two-minute exposures were madeand units for the regions of interest (ROI) were expressed asphotons/sec/cm2/sr. The blood was analyzed by flow cytometry forpresence of human CD45⁺CD56⁺CD3⁻ NK cells. A second experiment wasperformed to verify reproducibility of data.

Statistical Analysis Grouped data were expressed as mean±standard errormean (SEM). Differences between two groups were analyzed by Student's ttest. Multiple comparisons were analyzed by paired one-way ANOVA withTukey correction. Analysis was carried out in Graphpad Prism software.

Example 2 Construction of 1615EpCAM TriKE

Synthesis and assembly of a hybrid polynucleotide encoding 1615EpCAMTriKE (SEQ ID NO:8) was accomplished using DNA shuffling and ligationtechniques. The fully-assembled 1615EpCAM polynucleotide has, from the5′ end to the 3′end, an NcoI restriction site; an ATG initiation codon;coding regions encoding the VH and VL regions of human CD16 (NM3E2)derived from a phage display library produced by McCall et al. (MolImmunol., 1999, 36:433-445), a 20 amino acid segment(PSGQAGAAASESLFVSNHAY; SEQ ID NO:3), modified IL-15N72D, a seven aminoacid linker (EASGGPE; SEQ ID NO:4), and the humanized anti-EPCAM scFvfrom the antibody MOC-31; and finally a XhoI restriction site. Theresulting 1914 bp NcoI/XhoI polynucleotide was spliced into the pET21cexpression vector under control of anisopropyl-β-D-thiogalactopyranoside (IPTG) inducible T7 promoter. DNAsequencing analysis (Biomedical Genomics Center, University ofMinnesota, Minn., USA) was used to verify that the ploynuclleotide wascorrect in sequence and had been cloned in frame. Other constructs usedin this study, were created in a similar manner but including codingregions for monospecific anti-CD16 scFv, and anti-EpCAM scFv.

Inclusion Body Isolation

Bacterial protein expression was performed with Escherichia coli strainBL21 (DE3) (Novagen, Madison, Wis., USA) by plasmid transformation.After overnight culture, bacteria were grown in 800 ml Luria brothcontaining 50 mg/ml kanamycin. Induction of gene expression occurredwhen culture media reached an optical density (OD) 600 of 0.65 with theaddition of IPTG (FischerBiotech, Fair Lawn, N.J., USA). Two hours afterinduction, bacteria were harvested (from 5 liters cultured media a 43 gbacterial pellet was isolated). Next, the pellet was homogenized in abuffer solution (50 mM Tris, 50 mM NaCl, and 5 mM EDTA pH 8.0),sonicated and centrifuged. Pellets were extracted with 0.3% sodiumdeoxycholate, 5% Triton X-100, 10% glycerin, 50 mmol/L Tris, 50 mmol/LNaCl, 5 mmol/L EDTA (pH 8.0) and washed (final pellet weight: 12.5 g).

Refolding and Purification

Refolding and purification was performed as previously described(Schmohl et al., 2016. Target Oncol. 11(3):353-361). Briefly, in orderto refold, proteins from inclusion bodies (IB) where dissolved at 20:1(mg wet weight/mL) in solubilization buffer (7 M GuanidineHydrochloride, 50 mM Tris, 50 mM NaCl, 5 mM EDTA and 50 mM DTT, pH 8.0).Following a one-hour incubation at 37° C., pellets were removed bycentrifugation. The supernatant was diluted (20-fold) with refoldingbuffer (50 mM Tris-HCl, 50 mM NaCl, 0.8 mM L-arginine, 20% glycerin, 5mM EDTA and 1 mM GSSG, pH 8.0) at 4° C. for two days. The buffer wasremoved by 10-fold dialysis against 20 mM Tris-HCl, pH 9.0 in 20 mMTris-HCl, pH 9.0 over four column volumes. SDS-PAGE analysis wasperformed to evaluate purity. The fusion proteins were stained withSimply Blue life Stain (Invitrogen, Carlsbad, Calif.). The size of theTriKE was about 68860 Da.

NK Cell Isolation and Purification

A histopaque gradient (Sigma-Aldrich, St. Louis, Mo., USA) and SEPMATEtubes (Stemcell Technologies, Inc., Vancouver, Canada) were used toisolate peripheral blood mononuclear cells (PBMCs) from adult blood(Memorial Blood Center, Minneapolis, Minn., USA) of healthy volunteersand to obtain enriched NK cells via negative selection using magneticbeads per the manufacturer's protocol (Stemcell Technologies, Inc.,Vancouver, Canada). Samples were obtained after informed consent and inaccordance with the University of Minnesota human subjects InstitutionalReview Board and the Declaration of Helsinki.

Tissue Culture

The following cell lines were obtained from the American Type CultureCollection: Breast cancer cell lines BT-474, SK-BR-3; prostate cancercell lines PC-3, DU145; head-and heck cancer cell lines UMSCC-11B, NA;ovarian cancer cell line SKOV-1; colon carcinoma cell line HT-29; lungcancer cell line Calu-3; Burkitts lymphoma cell line Daudi; acutemyeloid leukemia cell line HL-60; human glioblastoma cell line U87.Carcinoma and glioblastoma cell lines were grown in monolayers usingtissue flasks (Fogh et al., 1977. J Natl Cancer Inst 59:221-226), HL-60and Daudi cell lines (Klein et al., 1968. Cancer Res 28:1300-1310) weregrown in suspension. Cells were maintained in either RPMI 1640 (BT-474,SK-BR-3, PC-3, DU-145, HT-29, Daudi, HL60, Calu-3), DMEM (UMSCC-11B, NA,SK-OV-3, U87) supplemented with 10% fetal bovine serum and 2 mmol/LL-glutamine. In addition to the preceding supplements, BT-474 mediacontained 10 IU/mL insulin. Cells were incubated in a humidifiedconstant 37C^(°) atmosphere containing 5% CO₂. When cells were 90%confluent, they were passaged using trypsin-EDTA for detachment. Cellcounts were conducted using a standard hemacytometer. Only cells with aviability >95%, as determined by trypan blue exclusion, were used forexperiments.

Binding/Blocking Assay

To evaluate binding, 4×10⁵ of the respective cancer cells (BT-474, PC-3,UMSCC-11B, Calu-3, Daudi, U87) were washed and incubated in 4° C. with10 nM Fluorescein isothiocyate (FITC)-labeled anti-EpCAM scFv for 30minutes. For the blocking assay 200 nM FITC labeled 1615EpCAM TriKE wasadded to either 500 nM of anti-EpCAM scFv or an anti-CD22-CD19 scFvconstruct and was incubated for 30 minutes in 4° C. with HT-29 coloncarcinoma cells. After washing, staining intensity was evaluated with anLSRII flow cytometer (BD Biosciences, San Jose, Calif., USA).

CD107a Degranulation Assay

Flow cytometry assays measuring cytolytic degranulation via CD107aexpression and IFN-γ presence were performed previously reported(Gleason et al., 2012. Mol Cancer Ther 11:2674-2684). PBMCs wereincubated overnight (37° C., 5% CO₂) in RPMI 1640 supplemented with 10%fetal calf serum and with recombinant IL-12 10 ng/ml (PeproTech, RockyHill, N.J.) and IL-18 100 ng/ml (R&D Systems, Inc., Minneapolis, Minn.,USA) as a positive control. Cells were washed in 1×PBS, treated with 30nM of 1615EpCAM TriKE or other drugs and incubated for 10 minutes at 37°C. with 5% CO₂. FITC-conjugated anti-human CD107a monoclonal antibody(mAb) (LAMP-1) (BD Biosciences, San Jose, Calif.) was added and furtherincubated for one hour with respective target cells (BT-474, SK-BR-3,PC-3, DU-145, HT-29, HL60, UMSCC-11B, NA, SK-OV-3). GolgiStop (1:1500)(BD Biosciences, San Jose, Calif.) and GolgiPlug (1:1000) (BDBiosciences, San Jose, Calif.) were added and cells were furtherincubated for three hours. Cells were washed in 1×PBS and stained withPE/Cy7-conjugated anti-CD56 mAb, APC/Cy 7-conjugated anti-CD16 mAb andPE-CF594-conjugated anti-CD3 mAb (BioLegend, Inc., San Diego, Calif.),incubated for 15 minutes and then fixed in 2% paraformaldehyde. Thencells were prepared for intracellular stain using permeabilizationbuffer (BD Biosciences, San Jose, Calif.). Cells were incubated withPacific Blue-conjugated anti-human IFN-γ (BioLegend, Inc., San Diego,Calif.) for 20 minutes, washed and evaluated by FACS analysis using aLSRII flow cytometer (BD Biosciences, San Jose, Calif.). Forcompensation CompBead Plus Anti-Mouse Ig, κ/Negative Control (BSA)Compensation Plus (7.5 μm) particles (BD Biosciences, San Jose, Calif.)were used.

Chromium-51 Release Cytotoxicity Assay

HT-29 target cells were labeled for 1 hour with 1 μCi of ⁵¹Cr per 1×10⁵target cells at 37° C., 5% CO₂. Washing procedures were performed toremove excess ⁵¹Cr. Labeled target cells were added to the wells of96-well round-bottom plates (5×10³ cells). Resting effector NK cellstreated with 1615EpCAM TriKE, EpCAM16 BiKE or negative controls wereadded to the plates. E:T ratio ranged between 20:1 and 0.08:1. Theamount of ⁵¹Cr released, which corresponds to target cell death, wasmeasured by a gamma scintillation counter, and the percent target celllysis was calculated as follows: [(experimental lysis−spontaneouslysis)/(maximal lysis−spontaneous lysis)]×100. To determine maximallysis, ⁵¹Cr-labeled target cells were treated with 3% Triton X for fourhours.

Luminex

For analysis of chemokines and cytokines, purified NK cells from sixhealthy volunteers were co-incubated in 96 well plates for 24 hours withHT-29 colon carcinoma cells at a 2:1 E:T ratio and the respective drugin a concentration of 50 nM at 37° C., 5% CO₂. After a 24 hourincubation time, cells were centrifuged and supernatants were collectedand stored at −80° C. until being analyzed. GM-CSF, IL-6, IL-8 and TNF-α(R&D Systems, Inc., Minneapolis, Minn.) were determined using theLuminex system (MAGPIX, Luminex, Austin, Tex.). Values represent μg/mland were interpolated from standard curves of the recombinant humanproteins by using Xponent 4.2 software (Luminex, Austin, Tex.).

Proliferation and Viability Assays

PBMCs or enriched NK cells from healthy donors were labeled withCELLTRACE Violet Cell Proliferation Dye (Thermo Fisher Scientific,Waltham, Mass.) according to the manufacturer's protocol. Afterlabeling, cells were cultured with 50 nM concentrations of therespective drugs. Cells were harvested after seven days, stained forviability with Live/Dead reagent (Invitrogen, Carlsbad, Calif.) andsurface stained for anti-CD56 PE/Cy7 (BioLegend, Inc., San Diego,Calif., USA) and anti-CD3 PE-CF594 (BD Biosciences, San Jose, Calif.) togate on the viable CD3⁻CD56⁺ population. Data were analyzed with FlowJosoftware version 7.6.5. (FlowJo, LLC, Ashland, Oreg., USA).

Statistical Analyses

Data are presented as mean+/−standard deviation. Differences between twogroups were analyzed by Student's t test or one-way-ANOVA. Analysis andpresentation of data was done with GraphPad Prism 5 (GraphPad Software,Inc., La Jolla, Calif.).

Example 3 Construction of EF91(Llama Anti-Human IL16)-IL15-CD33

Synthesis and assembly of a hybrid polynucleotide encoding the TriKEEF91(llama anti-human IL16)-IL15-CD33 (SEQ ID NO:14) was accomplishedusing DNA shuffling and ligation techniques. The fully-assembledpolynucleotide has, from the 5′ end to the 3′end, an NcoI restrictionsite; an ATG initiation codon; coding regions encoding the the VH and VLregions of EF91 (llama anti-human IL16), a 20 amino acid segment(PSGQAGAAASESLFVSNHAY; SEQ ID NO:3), modified IL-15, a seven amino acidlinker (EASGGPE; SEQ ID NO:4), and the humanized anti-CD33 scFv; andfinally a XhoI restriction site. The resulting NcoI/XhoI polynucleotidewas spliced into the pET21d expression vector under control of anisopropyl-3-D-thiogalactopyranoside (IPTG) inducible T7 promoter.

Example 4

Construction of 1615 antiHIV

Since 1615x is a platform technology, it is also possible to useanti-viral scFvs that are or are not associated with cancer development.Synthesis and assembly of a hybrid polynucleotide encoding the TriKE1615antiHIV (SEQ ID NO: 19) was accomplished using DNA shuffling andligation techniques. The fully-assembled polynucleotide has, from the 5′end to the 3′end, an NcoI restriction site; an ATG initiation codon; theVH and VL regions of the anti-CD16 scFv, a 20 amino acid segment(PSGQAGAAASESLFVSNHAY; SEQ ID NO:3), modified IL-15, a seven amino acidlinker (EASGGPE; SEQ ID NO:4), and an anti-HIV scFv; and finally a XhoIrestriction site.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

SEQUENCE LISTING FREE TEXT SEQ ID NO: 1MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAMKCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFLQSFVHIVQMF INTSEASGGP EQVQLVQSGA EVKKPGSSVK VSCKASGYTF TDYNMHWVRQAPGQGLEWIG YIYPYNGGTG YNQKFKSKAT ITADESTNTA YMELSSLRSE DTAVYYCARGRPAMDYWGQG TLVTVSSGGG GSGGGGSGGG GSDIQMTQSP SSLSASVGDR VTITCRASESVDNYGISFMN WFQQKPGKAP KLLIYAASNQ GSGVPSRFSG SGSGTDFTLT ISSLQPDDFATYYCQQSKEV PWTFGQGTKV EIK SEQ ID NO: 2MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNMHWVRQAPGQGLEWIGY IYPYNGGTGY NQKFKSKATI TADESTNTAY MELSSLRSED TAVYYCARGRPAMDYWGQGT LVTVSSGGGG SGGGGSGGGG SDIQMTQSPS SLSASVGDRV TITCRASESVDNYGISFMNW FQQKPGKAPK LLIYAASNQG SGVPSRFSGS GSGTDFTLTI SSLQPDDFATYYCQQSKEVP WTFGQGTKVE IK SEQ ID NO: 3 PSGQAGAAAS ESLFVSNHAY SEQ ID NO: 4EASGGPE SEQ ID NO: 5MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLNWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIHDTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTSQVQLVQSGAEVKKPGS SVKVSCKASG YTFTDYNMHW VRQAPGQGLE WIGYIYPYNG GTGYNQKFKSKATITADEST NTAYMELSSL RSEDTAVYYC ARGRPAMDYW GQGTLVTVSS GGGGSGGGGSGGGGSDIQMT QSPSSLSASV GDRVTITCRA SESVDNYGIS FMNWFQQKPG KAPKLLIYAASNQGSGVPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQS KEVPWTFGQG TKVEIKSEQ ID NO: 6MENWVNVISD LKKIEDLIQS MHIDATLYTE SDVHPSCKVT AMKCFLLELQ VISLESGDASIHDTVENLII LANDSLSSNG NVTESGCKEC EELEEKNIKE FLQSFVHIVQ MFINTSPSGQAGAAASESLF VSNHAYEVQL VESGGGVVRP GGSLRLSCAA SGFTFDDYGM SWVRQAPGKGLEWVSGINWN GGSTGYADSV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARGRSLLFDYWGQGTLVT VSRGGGGSGG GGSGGGGSSE LTQDPAVSVA LGQTVRITCQ GDSLRSYYASWYQQKPGQAP VLVIYGKNNR PSGIPDRFSG SSSGNTASLT ITGAQAEDEA DYYCNSRDSSGNHVVFGGGT KLTVLEASGG PEQVQLVQSG AEVKKPGSSV KVSCKASGYT FTDYNMHWVRQAPGQGLEWI GYIYPYNGGT GYNQKFKSKA TITADESTNT AYMELSSLRS EDTAVYYCARGRPAMDYWGQ GTLVTVSSGG GGSGGGGSGG GGSDIQMTQS PSSLSASVGD RVTITCRASESVDNYGISFM NWFQQKPGKA PKLLIYAASN QGSGVPSRFS GSGSGTDFTL TISSLQPDDFATYYCQQSKE VPWTFGQGTK VEIK SEQ ID NO: 7MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNMHWVRQAPGQGLEWIGY IYPYNGGTGY NQKFKSKATI TADESTNTAY MELSSLRSED TAVYYCARGRPAMDYWGQGT LVTVSSGGGG SGGGGSGGGG SDIQMTQSPS SLSASVGDRV TITCRASESVDNYGISFMNW FQQKPGKAPK LLIYAASNQG SGVPSRFSGS GSGTDFTLTI SSLQPDDFATYYCQQSKEVP WTFGQGTKVE IKEASGGPEN WVNVISDLKK IEDLIQSMHI DATLYTESDVHPSCKVTAMK CFLLELQVIS LESGDASIHD TVENLIILAN DSLSSNGNVT ESGCKECEELEEKNIKEFLQ SFVHIVQMFI NTS SEQ ID NO: 8MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAMKCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFLQSFVHIVQMF INTSEASGGP EDIQMTQSPS SLSASVGDRV TITCRSTKSL LHSNGITYLYWYQQKPGKAP KLLIYQMSNL ASGVPSRFSS SGSGTDFTLT ISSLQPEDFA TYYCAQNLEIPRTFGQGTKV ELKRATPSHN SHQVPSAGGP TANSGTSGEV QLVQSGPGLV QPGGSVRISCAASGYTFTNY GMNWVKQAPG KGLEWMGWIN TYTGESTYAD SFKGRFTFSL DTSASAAYLQINSLRAEDTA VYYCARFAIK GDYWGQGTLL TVSS SEQ ID NO: 9MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAMKCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFLQSFVHIVQMF INTSEASGGP EDIQMTQSPS SLSASVGDRV TITCRSTKSL LHSNGITYLYWYQQKPGKAP KLLIYQMSNL ASGVPSRFSS SGSGTDFTLT ISSLQPEDFA TYYCAQNLEIPRTFGQGTKV ELKRATPSHN SHQVPSAGGP TANSGTSGEV QLVQSGPGLV QPGGSVRISCAASGYTFTNY GMNWVKQAPG KGLEWMGWIN TYTGESTYAD SFKGRFTFSL DTSASAAYLQINSLRAEDTA VYYCARFAIK GDYWGQGTLL TVSSEPKSSD KTHTSPPSPD IVLSQSPAIMSASPGEKVTI SCSASSSVSY MYWYQQKPGS SPKPWIYRTS NLASGVPARF SGSGSGTSYSLTISSMEAED AATYYCQQYH SYPPTFGAGT KLELKSSGGG GSGGGGGGSS RSSLEVKLVESGPELKKPGE TVKISCKASG YTFTDYSMHW VNQAPGKGLK WMGWINTETG EPSYADDFKGRFAFSLETSA STAYLQINNL KNEDTATYFC ATDYGDYFDY WGQGTTLTVS SAKTTPPSVT SSEQ ID NO: 10MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAMKCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFLQSFVHIVQMF INTSEASGGP EDIVLSQSPA IMSASPGEKV TISCSASSSV SYMYWYQQKPGSSPKPWIYR TSNLASGVPA RFSGSGSGTS YSLTISSMEA EDAATYYCQQ YHSYPPTFGAGTKLELKSSG GGGSGGGGGG SSRSSLEVKL VESGPELKKP GETVKISCKA SGYTFTDYSMHWVNQAPGKG LKWMGWINTE TGEPSYADDF KGRFAFSLET SASTAYLQIN NLKNEDTATYFCATDYGDYF DYWGQGTTLT VSS SEQ ID NO: 11MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAMKCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFLQSFVHIVQMF INTSEASGGP EQVQLVQSGA EVKKPGASVK VSCKASGYSF TGYTMNWVRQAPGQGLEWMG LITPYNGASS YNQKFRGKAT MTVDTSTSTV YMELSSLRSE DTAVYYCARGGYDGRGFDYW GQGTLVTVSS GGGGSGGGGS SGGGSDIQMT QSPSSLSASV GDRVTITCSASSSVSYMHWY QQKSGKAPKL LIYDTSKLAS GVPSRFSGSG SGTDFTLTIS SLQPEDFATYYCQQWSKHPL TFGQGTKLEI K SEQ ID NO: 12MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLEPKSSDKTHT SPPSPNWVNV ISDLKKIEDL IQSMHIDATL YTESDVHPSC KVTAMKCFLLELQVISLESG DASIHDTVEN LIILANDSLS SNGNVTESGC KECEELEEKN IKEFLQSFVHIVQMFINTSP SGQAGAAASE SLFVSNHAYD IQMTQSPSSL SASVGDRVTI TCRSTKSLLHSNGITYLYWY QQKPGKAPKL LIYQMSNLAS GVPSRFSSSG SGTDFTLTIS SLQPEDFATYYCAQNLEIPR TFGQGTKVEL KRATPSHNSH QVPSAGGPTA NSGTSGEASG GPEDIQMTQTTSSLSASLGD RVTISCRASQ DISNYLNWYQ QKPDGTVKLL IYYTSILHSG VPSRFSGSGSGTDYSLTISN LEQEDFATYF CQQGNTLPWT FGGGTKLEIK GSTSGSGKPG SGEGSTKGEVQLVESGGGLV KPGGSLKLSC AASGFAFSIY DMSWVRQTPE KRLEWVAYIS SGGGTTYYPDTVKGRFTISR DNAKNTLYLQ MSSLKSEDTA MYYCARHSGY GTHWGVLFAY WGQGTLVTVSAGGGGSDILL TQTPASLAVS LGQRATISCK ASQSVDYDGD SYLNWYQQIP GQPPKLLIYDASNLVSGIPP RFSGSGSGTD FTLNIHPVEK VDAATYHCQQ STEDPWTFGG GTKLEIKRGSTSGSGKPGSG EGSTKGQVQL QQSGAELVRP GSSVKISCKA SGYAFSSYWM NWVKQRPGQGLEWIGQIWPG DGDTNYNGKF KGKATLTADE SSSTAYMQLS SLASEDSAVY FCARRETTTVGRYYYAMDYW GQGTSVTVSS SEQ ID NO: 13MDIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQK PDGTVKLLIY YTSRLHSGVPSKFSGSGSGT DYTLTISNLE QEDIATYFCQ QGNTLPWTFA GGTKLEIKRG GGGSGGGGSGGGGSGGREVQ LVQSGAELVK PGATMKISCK ASGYSFTGYT MNWVKQSHGK NLEWMGLINPYKGVSTYNQK FKDKATLTVD TSTDTAYMEL LSLTSEDSAV YYCARSGYYG DSDWYFDVWGAGTTVTVSSP SGQAGAAASE SLFVSNHAYP TSSSTKKTQL QLEHLLLDLQ MILNGINNYKNPKLTRMLTF KFYMPKKATE LKHLQCLEEE LKPLEEVLNL AQSKNFHLRP RDLISNINVIVLELKGSETT FMCEYADETA TIVEFLNRWI TFCQSIISTL TEASGGPEDI QMTQSPSSLSASVGDRVTIT CRSTKSLLHS NGITYLYWYQ QKPGKAPKLL IYQMSNLASG VPSRFSSSGSGTDFTLTISS LQPEDFATYY CAQNLEIPRT FGQGTKVELK RATPSHNSHQ VPSAGGPTANSGTSGEVQLV QSGPGLVQPG GSVRISCAAS GYTFTNYGMN WVKQAPGKGL EWMGWINTYTGESTYADSFK GRFTFSLDTS ASAAYLQINS LRAEDTAVYY CARFAIKGDY WGQGTLLTVS SSEQ ID NO: 14MKWVTFISLL FLFSSAYSQV QLVESGGGLV QPGGSLRLSC AASGLTFSSY NMGWFRQAPGQGLEAVASIT WSGRDTFYAD SVKGRFTISR DNSKNTLYLQ MNSLRAEDTA VYYCAANPWPVAAPRSGTYW GQGTLVTVSS SGGGGSGGGG SGGGGSGGGG SGNWVNVISD LKKIEDLIQSMHIDATLYTE SDVHPSCKVT AMKCFLLELQ VISLESGDAS IHDTVENLII LANNSLSSNGNVTESGCKEC EELEEKNIKE FLQSFVHIVQ MFINTSGSTS GSGKPGSGEG STKGQVQLVQSGAEVKKPGS SVKVSCKASG YTFTDYNMHW VRQAPGQGLE WIGYIYPYNG GTGYNQKFKSKATITADEST NTAYMELSSL RSEDTAVYYC ARGRPAMDYW GQGTLVTVSS GGGGSGGGGSGGGGSDIQMT QSPSSLSASV GDRVTITCRA SESVDNYGIS FMNWFQQKPG KAPKLLIYAASNQGSGVPSR FSGSGSGTDF TLTISSLQPD DFATYYCQQS KEVPWTFGQG TKVEIKVDESEQ ID NO: 15NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIHDTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTSSEQ ID NO: 16MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAMKCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFLQSFVHIVQMF INTSEASGGP EAKVQLQESG PSLVQPSQRL SITCTVSGFS LISYGVHWVRQSPGKGLEWL GVIWRGGSTD YNAAFMSRLS ITKDNSKSQV FFKMNSLQAD DTAIYFCAKTLITTGYAMDY WGQGTTVTVS SGGGGSGGGG SGGGGSDIEL TQSPSSFSVS LGDRVTITCKASEDIYNRLA WYQQKPGNAP RLLISGATSL ETGVPSRFSG SGSGKDYTLS ITSLQTEDVATYYCQQYWST PTFGGGTKLE IKR SEQ ID NO: 17MDIVLSQSPA IMSASPGEKV TISCSASSSV SYMYWYQQKP GSSPKPWIYR TSNLASGVPARFSGSGSGTS YSLTISSMEA EDAATYYCQQ YHSYPPTFGA GTKLELKSSG GGGSGGGGGGSSRSSLEVKL VESGPELKKP GETVKISCKA SGYTFTDYSM HWVNQAPGKG LKWMGWINTETGEPSYADDF KGRFAFSLET SASTAYLQIN NLKNEDTATY FCATDYGDYF DYWGQGTTLT VSSSEQ ID NO: 18PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEEELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRWITFCQSIIST LT SEQ ID NO: 19MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTGYADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARG RSLLFDYWGQ GTLVTVSRGGGGSGGGGSGG GGSSELTQDP AVSVALGQTV RITCQGDSLR SYYASWYQQK PGQAPVLVIYGKNNRPSGIP DRFSGSSSGN TASLTITGAQ AEDEADYYCN SRDSSGNHVV FGGGTKLTVLPSGQAGAAAS ESLFVSNHAY NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAMKCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFLQSFVHIVQMF INTSEASGGP EMGWSCIILF LVATATGVHS QVRLSQSGGQ MKKPGDSMRISCRASGYEFI NCPINWIRLA PGKRPEWMGW MKPRHGAVSY ARQLQGRVTM TRDMYSETAFLELRSLTSDD TAVYFCTRGK YCTARDYYNW DFEHWGQGTP VTVSSASTKG PSVFPLAPSSKSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSSLGTQTYICNV NHKPSNTKVD KKVEPKSCDK SEQ ID NO: 20MKWVTFISLL FLFSSAYSQV QLVESGGGLV QPGGSLRLSC AASGLTFSSY NMGWFRQAPGQGLEAVASIT WSGRDTFYAD SVKGRFTISR DNSKNTLYLQ MNSLRAEDTA VYYCAANPWPVAAPRSGTYW GQGTLVTVSS

1. A compound comprising: an NK engaging domain comprising a moiety thatselectively binds to CD16 or NKG2c; an NK activating domain operablylinked to the NK engaging domain and comprising IL-15 or a functionalfragment thereof, and a targeting domain that selectively binds to atarget antigen and is operably linked to the NK activating domain andthe NK engaging domain. 2-69. (canceled)
 70. The compound of claim 1,wherein the CD16 comprises CD16a.
 71. The compound of claim 1, whereinthe tumor antigen is present on a tumor cell.
 72. The compound of claim1, wherein the tumor antigen is a B7 family member.
 73. The compound ofclaim 1, wherein the tumor antigen is a B7H family member.
 74. Thecompound of claim 1, wherein the NK engaging domain moiety comprises anantibody or a binding fragment thereof or a nanobody.
 75. The compoundof claim 74, wherein the antibody fragment comprises an scFv, a F(ab)2,or a Fab.
 76. The compound of claim 74, wherein the antibody or abinding fragment thereof or the nanobody is human or humanized.
 77. Thecompound of claim 74, wherein the antibody or a binding fragment thereofor the nanobody is camelid.
 78. The compound of claim 1, wherein theIL-15 comprises an amino acid sequence of SEQ ID NO: 15 or a functionalvariant thereof.
 79. The compound of claim 78, wherein the functionalvariant of IL-15 comprises an N72D or N72A amino acid substitution ascompared to SEQ ID NO:
 15. 80. The compound of claim 1, wherein thetargeting domain moiety comprises an antibody or a binding fragmentthereof or a nanobody.
 81. The compound of claim 80, wherein theantibody binding fragment comprises an scFv, a F(ab)2, or a Fab.
 82. Thecompound of claim 1, wherein the NK engaging domain comprises CD16, theNK activating domain comprises IL-15, and the targeting domainselectively binds to a B7 family member.
 83. The compound of claim 1,wherein the NK engaging domain comprises CD16, the NK activating domaincomprises IL-15, and the targeting domain selectively binds to a B7Hfamily member.
 84. The compound of claim 1, comprising at least oneflanking sequence linking two of the domains.
 85. The compound of claim84, further comprising a second flanking sequence linking the two linkeddomains with the third domain.
 86. The compound of claim 85, wherein theflanking sequences flank the NK activating domain.
 87. The compound ofclaim 85, wherein a first flanking sequence is C-terminal to the NKengaging domain and wherein a second flanking sequence is N-terminal tothe anti-tumor targeting domain.
 88. The compound of claim 1, furthercomprising a second targeting domain.
 89. The compound of claim 1,further comprising a second NK engaging domain.
 90. The compound ofclaim 1, further comprising a second NK activating domain.
 91. Acomposition comprising: the compound of claim 1; and a pharmaceuticallyacceptable carrier.
 92. A method comprising: administering to a subjectthe compound of claim 1 in an amount effective to induce NK-mediatedkilling of a target cell.
 93. The method of claim 92, wherein the targetcell is a cancer cell.
 94. A method for stimulating expansion of NKcells in vivo, the method comprising: administering to a subject anamount of the compound of claim 1 effective to stimulate expansion of NKcells in the subject.
 95. A method of treating cancer in a subject, themethod comprising: administering to the subject an amount of thecompound of claim 1 effective for treating the cancer.
 96. The method ofclaim 95, wherein the cancer comprises prostate cancer, lung cancer,colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidneycancer, renal cancer, oral cavity cancer, pharynx cancer, pancreascancer, uterine cancer, thyroid cancer, skin cancer, head and neckcancer, cervical cancer, ovarian cancer, or hematopoietic cancer. 97.The method of claim 95, further comprising administering the compositionprior to, simultaneously with, or following chemotherapy, surgicalresection of a tumor, or radiation therapy.
 98. The method of claim 97,wherein the chemotherapy comprises altretamine, amsacrine,L-asparaginase, colaspase, bleomycin, busulfan, capecitabine,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin,daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide,fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine,hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan,mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine,oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed,temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine,vincristine, vindesine, and vinorelbine.